smoothened


REGULATION

Patched regulates Smoothened trafficking using lipoprotein-derived lipids

Hedgehog (Hh) is a lipoprotein-borne ligand that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. When Hh is absent, its receptor Patched (Ptc) represses Smoothened (Smo) signaling by an unknown catalytic mechanism that correlates with reduced Smo levels on the basolateral membrane. Ptc contains a sterol-sensing domain (SSD) and is similar to the Niemann-Pick type C-1 protein, suggesting that Ptc might regulate lipid trafficking to repress Smo. However, no endogenous lipid regulators of Smo have yet been identified, nor has it ever been shown that Ptc actually controls lipid trafficking. This study shows that Drosophila Ptc recruits internalized lipoproteins to Ptc-positive endosomes and that its sterol-sensing domain regulates trafficking of both lipids and Smo from this compartment. Ptc utilizes lipids derived from lipoproteins to destabilize Smo on the basolateral membrane. It is proposed that Ptc normally regulates Smo degradation by changing the lipid composition of endosomes through which Smo passes, and that the presence of Hh on lipoproteins inhibits utilization of their lipids by Ptc (Khaliullina, 2009).

A central feature of the Hh pathway is repression of Smo signaling by Ptc when Hh is absent. How this repression functions at a mechanistic level is not understood. Ptc represses Smo at substoichiometric levels and the two proteins do not appear to be enriched at similar subcellular locations in the steady state in vivo. Repression of Smo by Ptc correlates with changes in Smo subcellular localization and decreased stability (Khaliullina, 2009). Models for Ptc-mediated repression have been proposed based on its SSD and on its sequence similarity to RND (resistance nodulation division) family of proton-driven transmembrane transporters. Membrane sterol levels can alter trafficking and/or the stability of other proteins with SSDs, thus it has been proposed that lipids might control the repressive activity of Ptc. Alternatively, based on its similarity to RND transporters, it has been proposed that Ptc might regulate Smo by modulating lipid trafficking. This is an attractive idea because Smo activity can be regulated by small lipophilic molecules. Although these are plausible models, there has as yet been no evidence to suggest that Ptc alters lipid trafficking or that any lipid alters Ptc function. Furthermore, if Ptc did regulate lipid transport to repress Smo, it is not clear where contact between Smo and Ptc-mobilized lipids would occur (Khaliullina, 2009).

This study shows that one or more lipids derived from the Drosophila lipoprotein particle Lipophorin (Lpp) are required for normal Ptc-mediated Smo destabilization. Ptc sequesters a small fraction of internalized Lpp in the Ptc-positive endosomal compartment and regulates lipid trafficking from this compartment via its SSD. Mutation of the Ptc SSD causes at least one Lpp-derived lipid, sterol, to accumulate in Ptc endosomes. Given that RND permeases have rather broad substrate specificities, Ptc SSD mutation may also perturb the trafficking of additional lipids from this compartment. Similar mutations in NPC1 alter endosomal trafficking of sphingolipids, as well as sterols (Khaliullina, 2009).

Although Ptc-dependent sterol trafficking is clearly dependent on its SSD, Ptc has been reported to reduce the accumulation of neutral lipid in a manner that does not depend on its SSD. Rather, this effect of Ptc appears to depend on its ability to sequester Lpp. This may suggest that Ptc-mediated sequestration of Lpp diverts these particles away from trafficking pathways that promote neutral lipid storage (Khaliullina, 2009).

Ptc is normally present in a small fraction of endosomes and, unlike NPC1, does not generally perturb cellular sterol trafficking when mutated. How could altered trafficking in the small subset of endosomes that contain Ptc affect Smo? This study show that mutation of the Ptc SSD not only alters lipid trafficking in Ptc endosomes, but also causes Smo to accumulate in this compartment (as well as on the basolateral membrane). Similar endosomal colocalization between Ptc and Smo has been observed in vertebrate tissue culture cells. This suggests that Smo normally traffics through Ptc endosomes, where it can be exposed to lipids that are mobilized by the Ptc SSD. Thus, Ptc may control the level of basolateral Smo by changing the balance of Smo degradation and recycling from Ptc endosomes. Lipid accumulation in endosomes of NPC1 mutant cells influences the activity of Rab7, Rab9 and Rab4, perturbing degradation and recycling. The lipid composition of Ptc-positive endosomes may exert similar, but much more specific, effects on Smo, altering the total levels of Smo protein (Khaliullina, 2009).

Which Lpp lipids does Ptc use to regulate Smo trafficking? Although sterols are present in Lpp and the Ptc SSD does regulate sterol trafficking from Ptc endosomes, the data do not support a role for bulk membrane sterol in Smo regulation. Membrane sterol can be reduced sixfold by dietary depletion without changing Smo levels on the basolateral membrane or other aspects of Hh signaling. Furthermore, liposome-mediated addition of ergosterol, the most abundant membrane sterol in Drosophila, does not reverse Smo accumulation caused by LppRNAi. However, the data do not rule out the possibility that signaling sterol derivatives that act at low concentrations might be responsible. Lpp lipid extracts are now being fractionated to identify this active molecule (Khaliullina, 2009).

Loss of Lpp reproduces only a subset of the effects of Ptc mutation on Smo signaling -- although it stabilizes Smo and causes Smo-dependent accumulation of full-length Ci155, it does not allow target gene activation. Similar uncoupling of Ci155 stability and target gene activation is seen in fused and dally mutants. It seems unlikely that 'activation' of Ci155 simply requires higher levels of Smo than Ci155 stabilization; in LppRNAi discs, Smo levels reach those that result in target gene activation in wild-type discs over a broader region than in wild-type discs -- nevertheless, the range of target gene activation is narrowed. Rather, these data suggest that, although Ci155 stabilization is regulated by the level of Smo on the basolateral membrane, its activation as a transcription factor requires a separate Smo-dependent signal. Similarly, in vertebrate cells, where Ptc regulates Smo trafficking to the primary cilium, it is clear that ciliary localization is insufficient for Smo signaling to activate transcription of target genes. Other G-protein-coupled receptors activate multiple signal transduction pathways in response to different ligands. Thus, Smo trafficking and Smo activation may also be controlled by different ligands. Although Lpp lipids regulate Smo trafficking, other lipids mobilized by Ptc could have additional effects on Smo signaling activity (Khaliullina, 2009).

The following model would be consistent with what is already known of Ptc-dependent Smo regulation and with the new observations. Ptc sequesters Lpp into Ptc-positive endosomes and regulates lipid trafficking in this compartment via its SSD. Smo that passes through Ptc endosomes can be targeted either for degradation or recycling, depending on Ptc-dependent modulation of endosomal lipid composition, and on other functions of the Ptc protein. Lipids derived from Lpp particles bias Smo trafficking towards degradation. The balance of degradation versus recycling affects total Smo levels on the basolateral membrane and its ability to stabilize Ci155 (Khaliullina, 2009).

The association of Hh with Lpp may do more than simply promote the release of Hh from the membrane. Hh is thought to bind to the extracellular loops of Ptc -- a region that is important for conferring substrate specificity in RND family transporters. The presence of Hh on lipoproteins may block Ptc-mediated mobilization of their lipids. Alternatively, increased Ptc degradation upon Hh binding may prevent Lpp sequestration and lipid mobilization. In either case, association with Lpp particles efficiently positions Hh to interfere with, or alter, the mobilization of Lpp lipid contents by Ptc, helping to alleviate Ptc-mediated Smo repression. Thus, Hh may signal, in part, by influencing the utilization of the lipoprotein particles on which it is carried (Khaliullina, 2009).

Role of lipid metabolism in smoothened derepression in hedgehog signaling

The binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. This study shows that Smo activation is dependent on the levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). Loss of STT4 kinase, which is required for the generation of PI4P, exhibits hh loss-of-function phenotypes, whereas loss of Sac1 phosphatase, which is required for the degradation of PI4P, results in hh gain-of-function phenotypes in multiple settings during Drosophila development. Furthermore, loss of Ptc function, which results in the activation of Hh pathway, also causes an increase in PI4P levels. Sac1 functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation. Taken together, these results suggest a model in which Ptc directly or indirectly functions to suppress the accumulation of PI4P. Binding of Hh to Ptc derepresses the levels of PI4P, which, in turn, promotes Smo activation (Yavari, 2010).

A major regulatory step in the modulation of Hedgehog signaling occurs at the level of the two multipass transmembrane proteins, Patched and Smoothened. Genetic and biochemical studies suggest that the ligand Hh binds Ptc and functions in its inactivation. This inhibitory step is critical for the activation of Smo, which transduces the signal intracelluarly to promote Hh target gene activation. The importance of this regulatory step is further underscored by the observation that the Ptc/Smo interaction is the most commonly disrupted step in cancers caused upon aberrant Hh signaling (Yavari, 2010).

This article shows that phospholipid metabolism plays an important role in the modulation of Hh signaling at the level of Ptc/Smo interaction. In particular, the results show that an increase in the level of PI4P by the inactivation of Sac1 phosphatase leads to Smo protein relocalization to the membrane and an increase in Hh signaling in multiple tissues during Drosophila development. Furthermore the kinase (STT4), which is required for the generation of PI4P, is also required for the proper transduction of Hh signaling as indicated by its effects on Hh target gene expression. PI4P accumulation in the cell is a hallmark of sac1 mutations and is also seen upon loss of ptc activity. Furthermore, in sac1 mutant tissue, both increased membrane localization of Smo and accumulation of PI4P were found, whereas reduction in the PI4P kinase function leads to an hh-like loss of function phenotype. These results establish that phospholipid metabolism provides a critical regulatory input in the modulation of Hh signaling (Yavari, 2010).

Recent studies have proposed that Smo activation requires an input from a nonprotein small molecule. Cholesterol and its derivatives (oxysterols) are likely candidates for the small molecules required directly or indirectly for Ptc inhibition or Smo activation, because they also promote the translocation of Smo to the cilium. Because oxysterols are known to bind to vesicular transport proteins that also interact with phospholipids, further studies on possible cooperation between these two lipid types could further shed light the mechanism of Smo activation (Yavari, 2010).

Inactivation of Smo by Ptc occurs in a catalytic fashion in that a small number of Ptc molecules can inactivate many more Smo molecules. The current results provide an explanation for this nonstoichiometric inhibitory mechanism. The finding that inactivation of Ptc increases PI4P suggests that Ptc normally functions in keeping PI4P levels low within a cell. This could be achieved either by the down-regulation of the STT4 kinase or by the up-regulation of the Sac1 phosphatase. It is less likely that Ptc modulates Sac1 activity because in vivo localization studies in multiple models system have shown that Sac1 is predominantly localized to the Golgi and, as a result of proximity arguments alone, it seems a more likely possibility that Ptc modulates PI4P levels by down-regulating the lipid kinase. In this model, during normal Hh signaling, binding of Hh to Ptc will relieve repression of the kinase by Ptc and cause an increase in PI4P. As with all genetic analysis in Drosophila, the results do not imply direct protein interactions; currently unknown transduction components could exist, and future biochemical analyses will reveal which, if any, of the interactions is direct. However, the genetic analysis does allow a proposal of how an increase in the levels of this lipid can activate Hh signaling. Studies from both flies and vertebrate model system have suggested that the localization of Smo protein to the plasma membrane is essential for the activation of the pathway, and studies in multiple model systems have shown that PI4P function is essential in the vesicular transport of cargo proteins from the Golgi to the plasma membrane (Skwarek, 2009). It is therefore proposed that Hh binding to Ptc releases inhibition of a lipid kinase such as STT4, resulting in high PI4P levels. This aids vesicular transport of Smo to the membrane and causes its activation. A schematic representing the genetic model that is consistent with past and present data is shown in the graphical abstract. The results using Shh-responsive mouse fibroblasts indicate that mammalian Hh signal transduction is dependent on the activity of the murine STT4 ortholog. Previous localization studies suggest the STT4 ortholog contributes to plasma membrane PI4P pools, an observation consistent with a conserved role for PI4P metabolites in the control of Smo by mammalian Ptc1 (Balla, 2005, Wong, 1997). The observation that RNAi against the mammalian PIK1 homolog, PI4III kinase α, also reduces Hh signal transduction could suggest it has diverged in function between flies and mammals. Alternatively, PI4P pools could be exchanged more readily between membrane-bound subcellular compartments and the cell surface in mammalian cells, making the removal of either of the PI4III kinases affect global availability of PI4P derivatives. In mammalian cells, Smo activation is associated with translocation of the molecule to the primary cilium, a ubiquitous microtubule-based cell surface protrusion. Given that Drosophila cells appear to lack primary cilia, it will be of interest to determine whether PI4III kinase activity is required for Smo translocation (Yavari, 2010).

Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. This study provides evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. Many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci (Ogden, 2006).

This work demonstrates that targeting the association between Smo and the Cos2 cargo domain functionally separates the known molecular markers of the Hh pathway into two distinct categories: those events dependent on a direct association between the Cos2 cargo domain and Smo and those not dependent on this direct association. The Hh-induced readouts requiring direct Smo-Cos2 association include Smo phosphorylation, stabilization, and translocation to the plasma membrane, which facilitate intermediate to high level activation of Ci. Hh-induced Fu and Cos2 hyperphosphorylation, Hedgehog signaling complex relocalization from vesicular membranes to the cytoplasm, and Ci stabilization do not appear to require a direct Smo-Cos2 cargo domain association. Thus, although Smo is necessary for all aspects of Hh signaling, only the molecular events grouped with Ci activation appear to require direct association between Cos2 and Smo. In vivo, carboxyl-terminal Smo binding domain expression is also capable of attenuating Hh signaling. This observation is consistent with in vitro observation that carboxyl-terminal Smo binding domain inhibits critical requirement(s) for pathway activation (Ogden, 2006).

A model has been proposed suggesting the existence of two independently regulated pools of the Hedghog signaling complex (HSC), one involved in pathway repression (HSC-R), and one involved in activation (HSC-A). HSC-R is dedicated to priming Ci for processing into the Ci75 transcriptional repressor, whereas HSC-A is dedicated to activation of stabilized Ci155 in response to Hh. this study provides evidence that the effects of these two HSCs can be functionally separated by specifically targeting the interaction between Smo and the Cos2 cargo domain. Moreover, distinct molecular markers were identified for each HSC. It is proposed that in HSC-R, the membrane vesicle tethered Cos2 functions as a scaffold to recruit protein kinase A, glycogen synthase kinase 3ß, and casein kinase I, which in turn phosphorylate Ci. Hyperphosphorylated Ci is then targeted to the proteasome by the F-box protein supernumerary limbs (Slimb), where it is converted into Ci75. In response to Hh, Fu and Cos2 are phosphorylated and dissociate from vesicular membranes and microtubules, which is suggested to result in the attenuation of HSC-R function. This allows for the subsequent accumulation of full-length Ci. The mechanism by which HSC-R function is inhibited by Hh-activated Smo is not clear but appears to require the carboxyl-terminal tail of Smo and, by this analysis, appears to occur independently of a direct Smo-Cos2 cargo domain association. However, the direct Cos2-Smo association is critical for regulation of HSC-A. In the absence of Hh, HSC-A is tethered to vesicular membranes, through Smo, where it is kept in an inactive state. In the presence of Hh, Cos2 bound directly to Smo acts as a scaffold for the phosphorylation of Smo by protein kinase A, glycogen synthase kinase 3ß, and casein kinase I. Phosphorylation of Smo triggers its stabilization and relocalization to the plasma membrane with HSC-A, where Ci is proposed to be activated. Thus, Cos2 plays a similar role in both HSC-R and HSC-A. In the former case, coupling protein kinase A, glycogen synthase kinase 3ß, and casein kinase I with Ci and, in the latter case, coupling the same protein kinases with the carboxyl-terminal tail of Smo (Ogden, 2006).

An alternative interpretation of these data is that disruption of the Cos2 cargo domain-Smo association separates high and low level Hh signaling. It has been suggested that a second, low affinity Smo binding domain may reside within the coiled-coil domain of Cos2. Thus, high level signaling, where all aspects of the Hh pathway are activated may require both Cos2 interaction domains to be directly bound to Smo. In either scenario, HSC-R function would be regulated independently of HSC-A function (Ogden, 2006).

It is concluded that targeted disruption of Cos2 cargo domain-Smo binding by CSBD is able to functionally separate the activities ascribed to the two HSC model. This two-switch system is amenable to the formation of a gradient of Hh signaling activity across a field of cells, in that the relative activity of HSC-R to HSC-A is directly proportional to the level of Hh stimulation a cell receives. The opposing functional effects of the two complexes can then establish unique ratios of Ci75 to activated Ci, resulting in distinct levels of pathway activation on a per cell basis (Ogden, 2006).

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. Gprk2 function is required for transducing the Smo signal, and when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary (Molnar, 2007).

Drosophila GPRK2 was compared to members of the three mammalian GPRK subfamilies in humans and the highest amino acid identity was found in the kinase catalytic domain, with lower levels of similarity in the flanking N-terminal and C-terminal regions. GPRK2 was most similar to human GPRK4, with 87% identity in the kinase domain and 45% and 47% identity in the N- and C-terminal regions, respectively. GPRK2 displays substantially less sequence identity to mammalian GPRK3, which is necessary to desensitize odorant receptors (Peppel, 1997). The N-terminal region of GPRK2 includes a unique stretch of amino acids (Gly124-Gly261, 138 amino acids) containing asparagine-rich and glycine-rich clusters. This unique amino acid region is not present in other GPRK subfamilies and is not homologous to sequences in other proteins, on the basis of Basic Local Alignment and Search Tool (BLAST) searches. These data suggest that GPRK2 is a member of the GPRK4 family from mammals but may have additional functions compared to other GPRK4 family members (Tanoue, 2008).

Smo is the key transducer of a conserved signaling pathway regulating many developmental processes in vertebrates and invertebrates. The transmembrane protein Patched (Ptc) is the receptor for the ligand Hedgehog (Hh) and represses Smo activity in the absence of ligand. The binding of Hh to Ptc relieves this repression and allows Smo to signal to a protein complex that includes the transcription factor Ci/Gli. Smo controls the activation of Ci in the presence of the Hh ligand in part by preventing Ci proteolytic processing into a transcriptional repressor. In the Drosophila wing disc, the epithelium giving rise to the wing and thorax of the fly, Smo signaling controls the expression of several genes in anterior cells close to the anterior-posterior (A/P) compartment boundary and promotes the growth and patterning of the wing (Molnar, 2007).

The cytoplasmic tail of Drosophila Smo is a target for phosphorylation by protein kinase A and casein kinase I, and it has been shown that Smo phosphorylation by these kinases is essential for its activity and membrane accumulation. However, most of these phosphorylated residues are not conserved in its vertebrate counterparts. Recently, the G protein-coupled receptor kinase 2 (Grk2) has been shown to phosphorylate mammalian Smo (Chen, 2004). G protein-coupled receptor kinases (GRKs) selectively phosphorylate the ligand-activated form of G protein-coupled receptors (Lefkowitz, 2005). This phosphorylation promotes uncoupling from G proteins and also the recruitment of β-arrestins, which target the receptor for clathrin-mediated endocytosis. In addition, GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. There are two Drosophila GRKs, GPRK1 and GPRK2. GPRK1 modulates the amplitude of the visual response acting as a Rhodopsin kinase, whereas GPRK2 regulates the level of cAMP during Drosophila oogenesis (Schneider, 2005; Lannutti, 2001). Phosphorylation of mammalian Smo by GRK2 promotes its endocytosis in clathrin-coated pits in a process dependent on β-arrestin2 (Chen, 2004). However, whether this form of Smo internalization is part of a desensitization mechanism, as is the case for different G protein-coupled receptors (Lefkowitz, 2005), or if it participates in Hh signaling is still not known. To address the participation of GRKs during Smo signaling in Drosophila, the function was analyzed of Gprk2 during imaginal wing disc development. It was found that Gprk2 activity is required for Smo activation. Thus, the reduction of Gprk2 expression by interference RNA, or its elimination by a genetic mutation, causes the accumulation of Smo in wing disc anterior cells exposed to Hh. The accumulation of Smo is, however, correlated with reduced activity, because Smo high-level targets are not correctly activated and flies expressing Gprk2-RNAi display Hh loss-of-function phenotypes. Interestingly, the reduction in Gprk2 expression is able to antagonize the activity of Smo mutant forms that mimic its phosphorylation by protein kinase A and casein kinase 1, suggesting that additional phosphorylation by Gprk2 is a necessary step to obtain the correct activation of Smo to promote the expression of its targets requiring high levels of signaling (Molnar, 2007).

The expression of Gprk2 mRNA in the wing disc is generalized but appears increased in a stripe of cells located close to the A/P compartment boundary. To better characterize this pattern, the P-lacZ insertion Gprk206936, which is localized in the 5' untranslated region of the gene, was used. Interestingly, β-gal expression is restricted to the A/P compartment boundary of the wing disc during the third larval instar. The cells expressing β-gal were further identified by using a combination of region-specific markers such as Engrailed (En), Patched (Ptc), Blistered (Bs), and Caupolican (Caup). This analysis places the stripe of maximal expression of Gprk2 to anterior cells abutting the A/P boundary. These cells express Ptc and En in the anterior compartment and are localized in the region exposed to high-level Hh signaling. In fact, Hh signaling regulates the expression of Gprk2 in these anterior cells, because β-gal expression in Gprk206936 discs is expanded to the entire anterior compartment when hh is ectopically expressed, and it is repressed when the activity of the pathway is reduced by ectopic expression of Ptc). The regulation of Gprk2 accumulation in anterior cells by Hh suggests that Hh signaling and Gprk2 might be functionally related (Molnar, 2007).

All available Gprk2 alleles are P element insertions in the 5' region of the gene. These alleles are homozygous viable, and the mutant wings do not display any visible phenotype. Stronger loss-of-function conditions of the gene were generated by (1) expressing Gprk2 interference RNA (Gprk2i) under the control of yeast upstream activator sequences (UAS; UAS-Gprk2i) and (2) constructing a synthetic deletion of the gene. In wing discs of the combination Gal4-638/UAS-Gprk2i, a reduction of Gprk2 mRNA levels of 66% was found. The corresponding adult wings show a range of striking phenotypes similar to loss of Hh function, displaying a reduction of the L3/L4 intervein, fusion of the L3 and L4 veins, and in a lower percentage of wings, the loss of the L3 and L4 veins. These veins and the L3/L4 intervein correspond to the territory specified by Hh signaling. In fact, reduction of Gprk2 levels results in wings very similar to those with a moderate loss of Hh signaling, generated either by ectopic expression of Ptc or by expression or hh-interference RNA. This phenotype is very different from that observed upon increased activity of the pathway. The Gal4-638 line is expressed in the entire wing, and to distinguish between the effects of lowering Gprk2 levels in cells producing or responding to Hh, three other Gal4 lines were used expressed in either the anterior (Gal4-Ci and Gal4-ptc) or the posterior (Gal4-hh) compartments. It was found that the expression of Gprk2i only in anterior cells recapitulates the reduction of the L3/L4 intervein observed in Gal4-638/UAS-Gprk2i wings. Thus, the combinations Gal4-Ci/UAS-Gprk2i and Gal4-ptc/UAS-Gprk2i show a reduction or elimination of the L3/L4 intervein, whereas the wings of the Gal4-hh/UAS-Gprk2i combination display a normal pattern of veins. The phenotypes observed upon a reduction of Gprk2 unambiguously indicate that Gprk2 function is necessary for the transduction of the Hh signal. Furthermore, when the expression of Gprk2 is reduced in flies expressing lower levels of the ligand Hh, the resulting wings have stronger hh loss-of-function phenotypes, and a previously unrecognized phenotypic class indistinguishable to those of wings formed by smo mutant cells is now observed (Molnar, 2007).

To directly monitor the activity of the Hh pathway, the expression of several Hh targets was studied in Gal4-638/UAS-Gprk2i discs. The expression of En and Ptc in anterior cells is always impaired when Gprk2 levels are reduced. These two genes correspond to Hh targets activated by a high level of signaling. The expression of Knot (Kn) is also reduced in Gal4-638/UAS-Gprk2i discs, and the stripe of maximal accumulation of Ci is also modified in Gal4-638/UAS-Gprk2i discs compared with wild-type ones. The expression of other genes regulated directly or indirectly by Hh signaling was studied in Gal4-638/UAS-Gprk2i discs. The expression of the Notch ligand Delta (Dl) is very weak or absent in the primordia of the veins L3 and L4, where it accumulates at high levels in normal discs. Similarly, the expression of Bs in the L3/L4 intervein is reduced or absent in Gal4-638/UAS-Gprk2i discs. The expression of the low-level Hh signaling targets caup and decapentaplegic (dpp) is also modified in Gal4-638/UAS-Gprk2i discs. Caup expression in the presumptive L3 vein is generally expanded toward the A/P compartment boundary in Gal4-638/UAS-Gprk2i discs, most likely because En, a repressor of Caup in anterior cells, is not expressed upon a reduction of Gprk2 levels. The domain of Caup expression in the L3 vein is reduced or lost compared with wild-type discs in only a small fraction of discs (7%). The expression of dpp is detected in Gal4-638/UAS-Gprk2i discs at lower levels but in a domain broader than the characteristic of normal discs. Taken together, these data suggest that Gprk2 plays a positive role in the Hh signaling pathway. The lowering of Gprk2 levels reduces very efficiently high-level Hh signaling and much less efficiently low-level Hh signaling. Thus, a complete elimination of Hh signaling is observed only when Gprk2 levels are reduced in wing discs with lower hh. Finally, the expression of spalt, a target of the Dpp/BMP4 pathway, is almost normal upon Gprk2 reduction, indicating specificity of Gprk2 function toward Hh signaling (Molnar, 2007).

To confirm the specificity of the Gprk2 RNAi, the expression of two Hh-targets, En and Ptc, was analyzed in wing disc cells homozygous for a deficiency that removes all of the Gprk2 coding region. In both cases it was found that anterior Gprk2 - clones eliminate, in a cell-autonomous manner, the anterior expression of En. Gprk2 - clones located in the posterior compartment did not affect the expression of En, confirming that Gprk2 activity is required in cells receiving Hh (Molnar, 2007).

To further analyze where Gprk2 function is required in the Hh signaling pathway, the expression of En was studied in clones of cells ectopically expressing hh or both hh and Gprk2i. It was found that clones expressing Gprk2i located in the domain of En expression in the anterior compartment cell-autonomously suppress the expression of En. The expression of En is induced by Hh signaling in hh-expressing clones, both within the clone and in the surrounding cells. However, in the hh+Gprk2i-expressing clones, the expression of En is induced only in wild-type anterior cells that do not express Gprk2i. These observations confirm that Gprk2 activity is required for transducing the Hh signal in Hh-receiving cells and not for Hh secretion (Molnar, 2007).

Experiments in mammalian cells in culture have shown that beta-arrestin2 and GRK2 mediate internalization of active Smo (Chen, 2004). Consequently, the expression and subcellular localization of Smo was studied in wing discs where Gprk2 activity is reduced. In wild-type discs, smo RNA is expressed in all cells, but Smo protein accumulates associated to cell membranes only in the posterior compartment and in some anterior cells exposed to Hh. Intriguingly, the reduction in Gprk2 levels in the entire wing blade eliminates the distinction in Smo accumulation between anterior and posterior cells, and Smo is detected at similar levels in both compartments. When the levels of Gprk2 are reduced only in the dorsal compartment or in clones of Gprk2 - homozygous cells, the changes in Smo expression in anterior cells are more evident. Thus, it was observed that Smo accumulates at high levels associated to cell membranes in a broader anterior domain of cells within the range of Hh. The extension of Smo accumulation in anterior cells might be due to an extension of the Hh diffusion range because Ptc is not expressed in Gprk2 mutant cells. This is a previously unrecognized instance in which Smo accumulation and signaling can be uncoupled, because it was thought that, at least in Drosophila, Smo membrane accumulation leads to signaling. The same effects are observed when S2 cells were used. Thus, Smo is expressed in S2 cells in intracellular vesicles at low levels. Upon Hh treatment, Smo translocates close to the plasma membrane in these cells. In cells that have been treated for 4 days with Gprk2 dsRNA (causing a reduction of Gprk2 mRNA levels of 77%) the levels of Smo are higher independently of Hh (Molnar, 2007).

To further analyze the relationship between Smo and Gprk2 functions, Gprk2i was expressed in the same wing with different N-terminal (smoDeltaN; extracellular) and C-terminal (smoDeltaC2 and smoDeltaC4; intracellular) deletions of Smo. The expression of Smo proteins bearing either N-terminal or C-terminal deletions fails to rescue Smo mutants, but their overexpression does not interfere significantly with Smo signaling. A strong synergic genetic interaction was found when Smo C-terminal deletions were coexpressed with Gprk2i. Thus, wings expressing C-terminal deletions of Smo with reduced Gprk2 levels display a strong hh loss-of-function phenotype that is comparable to the elimination of smo. Gprk2i combined with UAS-smoDeltaN resulted in additive phenotypes. It is suggested that the reduction of Gprk2 uncovers a dominant-negative effect of SmoDeltaC proteins, reducing the efficiency of Smo signaling. The basis for this dominant negative effect could be the inclusion of a form of Smo, SmoDeltaC, unable to be phosphorylated by Gprk2, in the Smo complexes that have been postulated to mediate Smo activity. Therefore, it is proposed that Gprk2 function, acting through the C-terminal tail of Smo, is involved in an activation step promoting Smo interaction with the Costal2/Fused/Su(fu) complex to prevent Ci processing into a repressor form and to accumulate Ci in an activating form. Based on the effects of mammalian GRK2 and beta2-arrestin on Smo (Chen, 2004, Meloni, 2006), it is possible that Gprk2-mediated activation of Smo involves the recycling of Smo from the cell membrane to an intracellular signaling compartment (Molnar, 2007).

The interaction between SmoDeltaC and Gprk2 indicates a critical role of the Smo intracellular C-terminal domain for its relationship with Gprk2 function. Interestingly, the Smo intracellular C-terminal domain is where all of the consensus phosphorylation sites by casein kinase 1 and protein kinase A are located, as well as other serine and threonine residues in the vicinity of acidic residues that are similar to mammalian GRK2 phosphorylation consensus. A form of Smo was expressed in the wing disc that mimics its phosphorylation by these kinases (SmoSD123), and whether this Smo-activated form is sensitive to Gprk2 levels was analyzed. The expression of SmoSD123 in the wing disc causes overgrowth of the anterior compartment and defects in the L3 and L2 veins. In the corresponding wing discs, the accumulation of Smo and the expression of its targets En and Ptc are expanded to occupy the entire anterior compartment. When Gprk2 levels are reduced in discs expressing SmoSD123, Smo accumulation is still observed in all anterior cells. In contrast, the expression of both En and Ptc is now restricted to their normal domains adjacent to the A/P compartment boundary. The overgrowth phenotype characteristic of Gal4-638/+; UAS-SmoSD123 discs is not rescued by the reduction of Gprk2 expression, suggesting that the low-level Hh target dpp is still expressed through the anterior compartment. These data suggest that to generate the high levels of Smo activity required to activate the expression of its targets En and Ptc, the SmoSD123 protein has to be phosphorylated by Gprk2 (Molnar, 2007).

In conclusion, Drosophila Gprk2 is critically required to generate high levels of Hh signaling in the wing disc. The genetic interactions between Gprk2 and Smo proteins bearing C-terminal deletions or Smo phosphomimic variants suggest that Smo is a target of Gprk2. The modifications in Smo protein accumulation detected in wing discs and S2 cells with reduced Gprk2 expression suggests that a likely step affected by Gprk2 is the activation of Smo by a phosphorylation step that could prime Smo for internalization to a signaling compartment. GRK2 has recently been shown to play a positive role in Shh transduction in mammalian cells (Meloni, 2006). Taken together, these findings and the data indicate that Smo phosphorylation by GRK homologues constitute a conserved component of the Smo signal transduction cascade (Molnar, 2007).

Role of the Drosophila non-visual β-arrestin kurtz in hedgehog signalling

The non-visual β-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila, there is only a single non-visual β-arrestin, encoded by kurtz, whose function is essential for neuronal activity. This study addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, it was found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. These results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms (Molnar, 2011).

G-protein coupled receptors (GPCRs) are seven-transmembrane proteins that play critical roles during development and in the regulation of cellular physiology. GPCRs constitute the largest superfamily of cell membrane receptors. The major GPCR regulatory pathway involves phosphorylation of agonist-activated receptors by G protein-coupled receptor kinases (GRKs), followed by binding of the cytosolic arrestin proteins. This interaction prevents the receptor from activating additional G proteins in a process known as desensitization. GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. Thus, the GRK/β-arrestin pathway facilitates receptor internalization from the cell surface through clathrin-coated pits, and this leads to numerous physiological outcomes, including receptor degradation, receptor recycling and the activation of distinct downstream signalling events. Finally, more recent evidence suggest a role for β-arrestins in signalling by other families of cellular receptors, including receptor tyrosine kinase (RTKs), non-classical 7TMRs like Smoothened and Frizzled, Notch and TGFβ receptors, and also by downstream kinases such as MAPK and Akt/PI3K (Molnar, 2011).

The arrestin family is divided in two classes: the visual arrestins (arrestin 1 and 4), which are located almost exclusively in photoreceptor cells, and the non-visual β-arrestins 1 and 2 (also named arrestin 2 and 3, respectively), which are ubiquitously distributed. These proteins are closely related and their sequence is highly conserved across species. In Drosophila melanogaster there is only a single non-visual β-arrestin, encoded by kurtz (krz), which function is essential for development, survival and neural function. In addition, the gene CG32683 encodes a related protein that presents some homology with β-arrestins, but lacks the clathrin-binding domain. The GRK family includes seven members in humans (GRK1-7) and two components in flies (Gprk1 and Gprk2). Gprk1 modulates the amplitude of the visual response, acting as a Rhodopsin kinase, whereas Gprk2 regulates the level of cAMP during Drosophila oogenesis. In addition, Gprk2 and Gprk1 play a key role in the regulation of the Hedgehog (Hh) signal transduction pathway, where they seem to phosphorylate and activate the seven-pass transmembrane protein Smoothened (Smo). The β-arrestin Krz has also been involved in the regulation of Notch signalling, promoting the formation of a trimeric Notch-Deltex-Krz complex that mediates the degradation of the Notch receptor in an ubiquitination-dependent pathway, reminiscent of β-arrestin-mediated ubiquitination of other canonical GPCRs. More recently, Krz has also been implicated in the regulation of Smo accumulation and ERK phosphorylation. Because Krz is the unique β-arrestin present in Drosophila, it is likely that the protein has additional functions in the modulation of other signalling pathways (Molnar, 2011).

To address the participation of Krz in signalling events, its function during the development of the imaginal discs, the epithelial layers that give rise to the adult structures of the fly, was studied. Imaginal discs are very convenient model systems to study the activity of signalling pathways in vivo, because their development is under the regulation of the Hh, Wingless, EGFR, Notch, Insulin and TGFβ pathways. In this manner, the response of these epithelia to the manipulation of Krz levels using genetic variants is a key diagnostic to identify the functional requirements of this protein in signalling during imaginal development. Surprisingly, considering the key roles identified for vertebrate non-visual arrestins, this study found that the complete elimination of Krz in imaginal cells has no major consequences during imaginal development. Thus, and as claimed previously, krz mutant flies are morphologically normal. In contrast, the over-expression of Krz in the wing causes a phenotype identical to the loss of Hedgehog signalling. It was found that excess of Krz inhibits Hh signalling by promoting Smo internalization and degradation in a clathrin- and proteosomal- dependent manner. Contrary to that observed in vertebrates, the effects of Krz on Smo are independent of Gprk2 activity and of the activation state of the receptor. It is suggested that such differences in Hh signalling are based in the strict requirement of the primary cilia, a structure that is not present in fly epidermal cells, for Hh signalling in most vertebrates (Molnar, 2011).

This work has analysed the requirement of krz during the development of the Drosophila wing disc. The wing disc is an epithelial tissue, and its patterning and growth depends on the activity of several conserved signalling pathways. It was therefore reasoned that any requirement of Krz in the regulation of these pathways should be uncovered by the phenotype of the complete genetic loss of krz in the disc. Surprisingly, it was found that wing discs (and all other imaginal discs) can develop in an almost entirely normal manner in the total absence of Krz function. This finding implies that any role of Krz during normal development is dispensable for the regulation of the signalling pathways operating in the wing disc. It is emphasized that even small changes in the levels or domains of signalling by the Notch, EGFR and Hh/Smo pathways result in very characteristic and distinct phenotypes in the wing, and consequently it is concluded that these pathways operate normally in the absence of Krz in the discs (Molnar, 2011).

The function of Krz has been linked in imaginal discs with the regulation of Notch protein stability and of MAPK phosphorylation. These conclusions are base on sound biochemical data taken from cell culture experiments, and also on the analysis of genetic interactions evaluating the ability of krz mutations in heterozygosity to modify the phenotypes caused by Notch pathway components and MAPK alleles. It was also found that krz reduction enhances the phenotype of a Notch loss-of-function condition, no Notch-related phenotype was found in krz mutant wings. Furthermore, changes were found in Notch accumulation in a small fraction of krz1 and Df(3R)krz mutant clones, in contrast to. In this context, it is interesting to note that a robust accumulation of Notch was found when krz mutant cells over-express the Notch ligand Delta, suggesting that the function of krz becomes critical to promote Notch turnover upon Notch-Delta interactions. In this manner, the implication of this analysis and of previous works is that Krz might be required to optimise some aspects of Notch degradation or MAPK phosphorylation, but that these processes can occur normally in the absence of Krz. It might well be that only upon particular alterations of Notch levels, or in sensitized genetic backgrounds, such as over-expressing a non-dephosphorylable form of MAPK, these fine-tuning aspects of Krz are manifested in phenotypic modifications. It is unlikely that the paucity of krz requirements during imaginal development was due to functional redundancy with other arrestin proteins, because the only Drosophila candidate, CG32683, is not expressed in imaginal discs and does not affect imaginal development when over-expressed (Molnar, 2011).

The lack of a krz mutant phenotype in the discs is also surprising considering the multitude of roles assigned to its vertebrate counterparts in the Wnt, IGF, Notch, Smo and TGFβ signalling pathways and in ERK activation promoted by many GPCRs. These roles rely both on the regulation by β-arrestins of receptor internalization and subcellular localization, and also on their functions as scaffold for a variety of proteins involved in cellular signalling. It has to be postulated that insect epithelial cells have evolved arrestin-independent mechanisms to control receptor turnover and signalling, and consequently that arrestin function has become less relevant in these cells. This proposal is compatible with Krz retaining the capability to molecularly interact with similar proteins as its vertebrate counterparts, as Krz possesses both amino- and carboxy-terminal arrestin domains and is 72% similar to the mammalian β-arrestin 2 and 74% similar to β-arrestin 1 (Molnar, 2011).

In contrast to the loss-of-function analysis of krz, the study of its over-expression offers clear-cut indications of its implication in regulating Smo internalization. Thus, over-expression of Krz causes a very specific phenotype of loss-of-Hh signalling, manifested in defects localised in the central part of the wing that in extreme cases lead to the total failure of wing development. These phenotypes are associated to the loss of expression of Hh target genes, confirming that they are caused by reduced Hh signalling. As previously described, increased levels of Krz are extremely effective in reducing Smo accumulation in the cell membrane (Cheng, 2010 and this work). This effect is observed with wild type forms of Smo, with Smo mutated in its phosphorylation sites and with a phospho-mimic Smo protein that is constitutively activated. The elimination of Smo is also observed in posterior cells, indicating that Krz promotes Smo elimination independently of Ptc, and also in anterior cells localised away from the source of Hh, suggesting that Krz affects Smo turnover in the absence of ligand. Finally, the elimination of Smo by excess of Krz is independent of Gprk2 activity, because it is still observed in cells deficient for the Gprk2 gene. Gprk2 is required for the transduction of Smo signal, and when Gprk2 levels are lowered, inactive Smo accumulates at the cell membrane (Molnar, 2010). In the double combination (excess of Krz plus loss of Gprk2), Smo is eliminated, suggesting that Smo unmodified by Gprk2 is still capable to interacting with Krz and being removed. The resulting flies show extreme hh loss-of-function phenotypes, likely the result of both loss of Gprk2-dependent Smo activation and increased, Krz-promoted, Smo turnover (Molnar, 2011).

The ability of Krz to interact with Smo in the Drosophila wing is very specific, since no other alterations in the localization and activity of other receptors, such as Notch or EGFR. In addition, β-arrestin 2 promotes, upon GRK phosphorylation, the internalization of activated Smo in human embryonic kidney 293 cells. Finally, β-arrestin 2 promotes Smo signalling in zebrafish embryos, and this seems to be a physiological function because it is detected in loss-of-function conditions. In contrast, a clear antagonism of Krz on Smo signalling caused by Smo internalization and degradation promoted was observed only by excess of Krz, and this effect of Krz is independent of the Smo phosphorylation state and of Gprk2 activity (Molnar, 2011).

One of the main differences in the Smo signalling pathway between vertebrates and Drosophila is the localization in vertebrates of active Smo to the primary cilium, a structure that is present only in the fly in sensory neurons. It can only be speculated that the necessity to translocate Smo complexes associated with the type II kinesin motor Kif3A to the cilium, a structure not present in fly epidermal cells, imposes a requirement for β-arrestins that is not observed in the fly. Nonetheless, the current results show that the capability of Krz to interact with Smo is retained in Drosophila, and this is revealed upon the over-expression of Krz. Once Krz is bound to Smo it would trigger the formation of clathrin-coated pits that targets Smo for degradation in the proteasome, leading to the insufficiency of Hh signalling that was observed. In this way, it is proposed that Krz has retained some of the molecular targets typical of vertebrate β-arrestins, but that these interactions might not occur at physiological levels of expression, or being redundant with other mechanisms of receptor trafficking and signalling (Molnar, 2011).

Control of antagonistic components of the Hedgehog signaling pathway by microRNAs in Drosophila

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. Stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. This study shows that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs (Friggi-Grelin, 2009).

cos2, fu, and smo mRNA can be regulated by a cluster of microRNAs, including miR-12 and miR-283, in Drosophila wing disc. The overexpression of this cluster decreases the levels of Smo, Cos2, and Fu proteins and activates the Hh pathway, as evidenced by the induction of dpp expression in the wing imaginal discs and by the adult wing outgrowth. The experiments presented in this study with the 3'-UTR sensors of smo, fu, or cos2 are in favor of a direct binding. To constitute a real proof of a direct effect, further experiments as direct biochemical binding assay or compensatory mutation between the 3'-UTR and the miRNAs will be necessary to perform (Friggi-Grelin, 2009).

Programs that have been created to genomewide predictions of Drosophila miRNA targets provide lists of presumptive miR-12, and miR-283 regulated genes. In addition to the current in vivo validations, miR-12 binding sites are predicted on the 3'-UTR of ci and no sites were found on the 3'-UTR of the Su(fu) gene. No decrease was observed in either of these two proteins in the microRNA cluster overexpressing clones. It is interesting to note that Su(fu) mRNA, encoding another negative regulator of Hedgehog signaling, has been shown to be targeted by miR-214 in zebrafish. Absence of miR-214 results in the reduction of muscle cell types, the specification of which is dependent on Hh pathway activity. Nevertheless, the current study shows that in Drosophila wing discs an absence of microRNA does not modify the Hh pathway, raising the question of what the role of microRNAs in Drosophila Hh pathway regulation is (Friggi-Grelin, 2009).

Could the microRNAs overexpression phenotype that was identified be artifactual and simply the result of forced overexpression of the microRNA cluster in a tissue in which it should be silent? It is thought that the answer is no, because Northern blot analysis and the increase of miR-sensor in the dcr-1 mutant clones showed that the microRNA cluster is indeed expressed in this tissue. This suggests that the cluster likely has a role in this tissue in which it is normally present. Is the microRNA cluster regulation of the cos2 and smo 3'-UTRs physiological? It is thought so, because an absence of either the microRNA cluster or of Dicer in the wing imaginal disc induces an increase in the Cos2- and Smo-sensor lines. This signifies that the microRNAs expressed from the cluster regulate the cos2 and smo 3'-UTRs and thus display some functionality in the disc during larval development. Altogether, these data clearly show that an artifactual situation in which the microRNA cluster is expressed in a tissue in which it should not be present has not been created. The miRs overexpression was also tested on embryonic patterning but it did not lead to any phenotype, suggesting that the miR cluster regulation on the Hh pathway is specific to larval tissues (Friggi-Grelin, 2009).

As miR-12 and miR-283, and likely redundant miRs, are present in every cell of the wing disc, one possibility is that their normal roles are to dampen down the levels of Hh pathway components, particularly Cos2 and Smo, to prevent the accidental activation or downregulation of the pathway. Indeed, expressing both the microRNA cluster and its targets in the same tissue could provide a means of 'buffering stochastic fluctuations' in mRNA levels or in protein translation rates within the Hh signaling pathway, as has been proposed for other processes (Friggi-Grelin, 2009).

The data possibly indicate that miRNAs are able to regulate two antagonistic components of the pathway, Cos2 and Smo. It has been shown that the stability of these two proteins is 'interdependent': an increased level of Cos2 in the wing imaginal disc lowers the level of Smo, and, in the opposite direction, increased Smo decreases the level of Cos2. It is proposed that the interregulation of Cos2/Smo levels is independent of their relative activities because Cos2 effect on Smo levels is observed in posterior cells in which Cos2 activity is strongly inhibited by the constitutive activation of the pathway. Therefore, eliminating the miRNA-mediated inhibition of Cos2 and Smo in Delta3miR or dcr-1 mutant cells likely initially increased the levels of both proteins, but then the resulting higher levels of each protein presumably downregulated the other; the net variation of Cos2 and Smo levels would therefore be null. This hypothesis is favored because the independent Smo- and Cos2-sensor lines, which are unaffected by this Cos2/Smo interregulation, showed increased levels of GFP staining in Delta3miR and dcr-1 mutant animals. This suggests that the levels of both Cos2 and Smo are increased in the mutant animals but, because of the downregulation of each protein by the other, no ultimate alterations in the levels of the proteins are observed. If so, an Hh phenotype would not be expected to be seen in the miR mutant (Friggi-Grelin, 2009).

The screen created a situation in which the expression of the microRNA cluster is deregulated, ultimately destabilizing Cos2 protein levels and thereby activating Ci and Hh target gene expression. Importantly, a similar situation might be encountered during tumoral development. Aberrant Hh signaling activity is known to trigger the development of diverse cancers. While several of these tumors have been linked to mutations in Hh signaling components, not all of them have, leaving open the possibility that they are caused by other factors such as microRNA misexpression. Interestingly, more than half of the known human microRNA genes are located near chromosomal breakpoints associated with cancer, and in some documented cases the microRNAs are amplified, leading to overexpression. Some upregulated microRNAs are possibly able to bind mRNAs encoding negative regulators of Hh signaling, such as Su(fu) or Ptc, and could thus induce the misactivation of the Hh pathway, as is observed in some cancers. Therefore, a fine analysis of microRNA expression levels and the levels of known Hh components should be considered in studies of Hh pathway-related cancers (Friggi-Grelin, 2009).

What does this study add to the current knowledge about miRNA regulation? The study shows that a cluster of three microRNAs can target several antagonistic components of the same pathway in vivo. This is novel and unexpected. This raises the question of how to interpret the miRNA expression signatures observed in human tumors. Indeed, as stated above, it has been proposed that miRNAs are differentially expressed in human cancers and contribute to cancer development. The working hypothesis in the cancer/miRNAs field is that key cancer genes are regulated by aberrant expression of miRNAs. The identification of a specific miRNA:mRNA interactor pair is generally accepted as being of biological importance when the mRNA encodes a tumor suppressor or an oncogene whose expression is modified in the tumor. This study shows indirectly that this is an oversimplified view, because identifying an oncogene or tumor suppressor as a target of a miRNA may not provide a full explanation for tumor development if the same miRNA hits other antagonistic components of the same pathway that nullify the effect of the identified miRNA:mRNA interactor pair (Friggi-Grelin, 2009).

The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation

Hedgehog (Hh) transduces signal by regulating the subcellular localization and conformational state of the GPCR-like protein Smoothened (Smo) but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. This study shows that Hh-induced Smo conformational change recruits Costal2 (Cos2)/Fused (Fu) and promotes Fu kinase domain dimerization. Induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL) and phospho-mimetic mutations of AL activate the Hh pathway. Interestingly, it was observed that graded Hh signals progressively increase Fu kinase domain dimerization and AL phosphorylation, suggesting that Hh activates Fu in a dose-dependent manner. Moreover, it was found that activated Fu regulates Cubitus interruptus (Ci) by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. Evidence is provided that activated Fu exerts these regulations by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. Taken together, these results suggest that Hh-induced Smo conformational change facilitates the assembly of active Smo-Cos2-Fu signaling complexes that promote Fu kinase domain dimerization, phosphorylation and activation, and that Fu regulates both the activator and repressor forms of Ci (Shi, 2011).

How Hh signal is transduced from the GPCR-like receptor Smo to the transcription factor Ci/Gli is still poorly understood. A major unsolved issue is how a change in the Smo activation state is translated into a change in the activity of intracellular signaling complexes, which ultimately changes the balance between CiR/GliR and CiA/GliA. The current study suggests that Hh-induced conformational change of Smo exposes a Cos2 docking site(s) near the Smo C terminus that facilitates the assembly of an active Smo-Cos2-Fu complex, and that Smo activates Fu by promoting its kinase domain dimerization and phosphorylation. Evidence is provided that graded Hh signals progressively increase Fu kinase domain dimerization and phosphorylation, which may generate a Fu activity gradient, and that activated Fu regulates both CiR and CiA by controlling Ci-Sufu and Ci-Cos2-kinase complex formation (Shi, 2011).

Previous immunoprecipitation studies have revealed that Smo pulled down Cos2/Fu in both quiescent cells and Hh-stimulated cells, suggesting that Smo can form a complex with Cos2/Fu even in the absence of Hh. Furthermore, deletion analyses have indicated that both a membrane proximal domain and a C-terminal region of Smo C-tail can mediate the interaction between Smo and Cos2/Fu. Intriguingly, deleting the C-terminal region impaired, whereas deleting the membrane proximal domain potentiated, Smo activity in vivo. Further study suggested that the membrane proximal domain recruits Cos2/PP4 to inhibit Smo phosphorylation and cell-surface accumulation, which is released by Fu-mediated phosphorylation of Cos2 Ser572 in response to Hh. These observations suggest that Smo-Cos2-Fu interaction is likely to be dynamic and that distinct complexes may exist depending on the Hh signaling status. For example, Cos2 may associate with the membrane proximal region of Smo to inhibit Smo phosphorylation in quiescent cells. Upon Hh stimulation, Cos2/Fu may interact with the C-terminal region of Smo to transduce the Hh signal. In support of this model, it was found that Hh stimulated the recruitment of Cos2/Fu to the C-terminal region rather than the membrane proximal region of the Smo C tail. The increased binding depends on phosphorylation-induced conformational change of Smo C-tail that may expose the C-terminal Cos2 binding pocket(s) (Shi, 2011).

Hh signaling induces Fu kinase domain dimerization in a dose-dependent manner, most probably as a consequence of phosphorylation-induced conformational change and dimerization of Smo C tails. In addition, Hh-induced Fu dimerization depends on Cos2. Importantly, dimerization through the Fu kinase domain (CC-Fu) triggers Fu activation both in vitro and in vivo. Furthermore, CC-Fu can activate Ci in smo mutant clones and restore high levels of Hh signaling activity in cos2 mutant discs. Taken together, these results support a model in which Hh-induced Fu dimerization via Smo/Cos2 leads to Fu activation (Shi, 2011).

Both Fu dimerization and Hh stimulation induce phosphorylation of multiple Thr/Ser residues in the Fu activation loop that are important for Fu activation. Fu phosphorylation depends on its kinase activity and Fu can trans-phosphorylate itself, suggesting that Hh and dimerization may induce Fu autophosphorylation, although the results do not exclude the involvement of additional kinase(s). CC-induced dimerization does not fully activate Fu, suggesting that Smo may promote Fu activation through additional mechanisms. Activated Fu can promote phosphorylation of its C-terminal regulatory fragment, raising a possibility that Fu activation may also involve phosphorylation of its regulatory domain. Indeed, while this manuscript was under review, Zhou and Kalderon provided evidence that phosphorylation of several Ser/Thr residues in the Fu regulatory domain, likely by CK1, modulates the activity of an activated form of Fu (Zhou, 2011; Shi, 2011 and references therein).

The involvement of multiple phosphorylation events in Fu activation may provide a mechanism for fine-tuning Fu activity in response to different levels of Hh. Indeed, the efficiency of Fu dimerization and the level of activation loop phosphorylation correlate with the level of Hh signaling. Furthermore, the level of Fu activity correlates with the level of its activation loop phosphorylation. Thus, graded Hh signals may generate a Fu activity gradient by progressively increasing its dimerization and phosphorylation in response to a gradual increase in Smo phosphorylation and C-tail dimerization (Shi, 2011).

The conventional view is that Fu is required for high levels of Hh signaling by converting CiF into CiA. In support of this notion, fu mutations only affect the high, but not low, threshold Hh responsive genes. However, Fu function could have been underestimated because none of the fu mutations examined so far represents a null mutation. In addition, the existence of paralleled mechanisms, such as Gαi activation, could mask the contribution of Fu to low levels of Hh signaling. Nevertheless, a recent study using the phospho-specific antibody against Cos2 Ser572 revealed that Fu kinase activity could be induced by low levels of Hh, raising an interesting possibility that Fu may contribute to all levels of Hh signaling (Raisin, 2010). However, the lack of a fu-null mutation and the involvement of Fu in promoting Ci processing, probably through a structural role, make it difficult to directly demonstrate a role of Fu in blocking Ci processing. Using an in vivo assay for Ci processing, it was demonstrated that activated forms of Fu block Ci processing into CiR. In addition, this study found that activated Fu attenuates the association between Cos2 and Ci, as well as their association with PKA/CK1/GSK3, probably by phosphorylating Cos2, suggesting that activated Fu may block Ci processing by impeding the formation of the kinase complex required for efficient Ci phosphorylation (Shi, 2011).

Evidence is provided that activated Fu attenuates Ci/Sufu interaction. Because Sufu impedes Ci nuclear localization and may recruit a co-repressor(s) to further inhibit Ci activity in the nucleus, dissociation of Ci from Sufu may lead to the conversion of CiF to CiA. Interestingly, recent studies using mammalian cultured cells revealed that Shh signaling induces dissociation of full-length Gli proteins from Sufu (Humke, 2010; Tukachinsky, 2010), suggesting that inhibition of Sufu-Ci/Gli complex formation could be a conserved mechanism for Ci/Gli activation. Although activated forms of Fu promote Sufu phosphorylation, phospho-deficient and phospho-mimetic forms of Sufu behaved in a similar manner to wild-type Sufu in functional assays (Zhou, 2011), implying that phosphorylation of Sufu might not be a major mechanism through which Fu activates Ci. It has been shown that Shh also induces phosphorylation of full-length Gli3 that correlates with its nuclear localization (Humke, 2010). Furthermore, a Fu-related kinase Ulk3 can phosphorylate Gli proteins and promote their transcriptional activities (Maloverjan, 2010). Thus, Fu may activate Ci by promoting its phosphorylation, an interesting possibility that awaits further investigation (Shi, 2011).

Protein Interactions

Genetic and expression evidence point to a role of both Patched and Smoothened in Hedgehog receptor function. In wings bearing large anterior compartment mutant smo clones there appears to be an anterior shift in the distribution of dpp-exressing cells. This shift is interpred as evidence that the loss of smo activity abolishes the ability of anterior cells to respond to HH and also allows HH to spread abnormally far into the anterior compartment until it reaches, and is transduced by smo+ cells. One mutant of ptc, ptcS2 gives rise to a PTC protein that appears indistinguishable from null alleles when assayed for its ability to repress inappropriate activity of the HH signal transduction pathway. Nevertheless, ptcS2 retains an activity that allows anterior compartment cells to sequester HH. Additionally, up-regulation of ptc by HH, a conserved feature of HH signaling, is required to limit the movement of HH from the posterior into the anterior compartment. Finally, PTC represses the HH signal transduction pathway by blocking the intrinsic activity of SMO. This suggests that PTC is positioned upstream of SMO in the HH signal transduction pathway, either as a factor that regulates SMO-transducting activity in response to HH or as a factor that facilitates the direct modulation of SMO activity by HH (Chen, 1996).

During Drosophila development, cells belonging to the posterior compartment of each segment organize growth and patterning by secreting Hedgehog (Hh), a protein that induces a thin strip of adjacent cells in the anterior compartment to express the morphogens Decapentaplegic (Dpp) and Wingless (Wg). Hedgehog is bound and transduced by a receptor complex that includes Smoothened (Smo), a member of the Frizzled (Fz) family of seven-pass transmembrane receptors, as well as the multiple-pass transmembrane protein Patched (Ptc). Ptc is required for the binding of Hh to the complex as well as for the Hh-dependent activation of Smo within the complex. A likely null allele of the smo gene has been identified. It was used to determine whether Hh is bound by Ptc alone, or by Smo in concert with Ptc. Cells devoid of Smo can sequester Hh, but their ability to do so depends, as in wild-type cells, on the expression of high levels of Ptc protein. These results suggest that Ptc normally binds Hh without any help from Smo and hence favor a mechanism of signal transduction in which Hh binds specifically to Ptc and induces a conformational change leading to the release of latent Smo activity (Chen, 1998).

During wing development, cells in the posterior (P) compartment secrete Hh and cells in the anterior (A) compartment respond to Hh by turning on, or up-regulating, several genes, including those encoding Dpp and Ptc. These genes are expressed at high level only in a thin strip of A cells adjacent to the A/P compartment boundary, indicating that Hh normally moves only a short distance into the A compartment. Examined were clones of A compartment cells homozygous for the smo3 mutation, which codes for an altered protein that might not be null because it may retain the ability to insert into the cell membrane. These clones fail both to express dpp and to upregulate Ptc expression, even when adjacent to the A/P compartment boundary, indicating that they are unable to transduce Hh. Moreover, they appear unable to restrict the movement of Hh into the A compartment, as indicated by the response of wild-type cells positioned just anterior to large A compartment clones of smo3 cells that abut the A/P boundary. These smo+ cells express dpp and Ptc at high levels, even though they are located many cell diameters away from the A/P boundary, at positions that are normally too far from the boundary to be exposed to Hh. The same analysis was performed using the smoD16 allele, certain to be a null allele because it gives rise to a truncated protein, in place of the smo3 allele, and the same result was obtained. Both Ptc expression as well as the expression of a dpp-lacZ transgene, placed in trans to the smoD16 mutant allele, were monitored. A compartment cells lacking Smo protein not only fail to respond to Hh, but also fail to limit the further spread of Hh into the A compartment, indicating that they lack a Hh-sequestering activity (Chen, 1998).

In the case of the smo3 mutant allele, the failure of mutant cells to impede the movement of Hh into the A compartment can be attributed to their failure to up-regulate the expression of Ptc, which confers Hh-sequestering activity. Indeed, it is possible to restore the ability of smo3 mutant cells to restrict Hh movement by simultaneously eliminating the activity of Protein kinase A (PKA), a manipulation that constitutively activates the Hh signal transduction pathway causing smo3 mutant cells to express high levels of Ptc. However, it is not known whether the Hh-sequestering activity of smo3 PKA - cells is mediated solely by Ptc, or by smo3 mutant protein in conjunction with Ptc. Indeed, molecular analysis indicates the smo3 allele encodes a truncated form of Smo that includes the entire cysteine rich extracellular domain (CRD), as well as the first three transmembrane domains, consistent with the possibility that truncated Smo retains a Hh-binding activity. To resolve this uncertainty, the smo-;PKA- experiment was repeated using the smoD16 allele in place of the smo3 allele. smoD16;PKA- clones were generated using two genetic configurations. In the first configuration, both Ptc expression as well as the expression of a dpp-lacZ transgene were monitored. In contrast to smoD16 clones, which express only low levels of Ptc protein, smoD16;PKA- clones autonomously express high levels of Ptc protein throughout. Moreover, large smoD16;PKA- clones that abut the A/P boundary differ from similarly positioned smoD16 clones in that they appear to impede the movement of Hh through the clone. In the second genetic configuration, smoD16;PKA- mutant cells were marked by the loss of a ubiquitously expressed arm-lacZ transgene. This configuration allows the smo PKA genotype to be assessed independently of Ptc expression. A compartment cells that are homozygous for the smoD16 mutation and hence devoid of Smo protein are nevertheless able to sequester Hh, provided that they also express high levels of Ptc. These findings favor the proposal that Hh normally binds specifically to Ptc within the Hh receptor complex without any direct involvement of Smo (Chen, 1998).

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened and the transcription factor Cubitus interruptus (Ci). Protein kinase A (PKA) has been implicated in the mechanism of Hh signal transduction because it acts to repress Hh target genes in imaginal disc cells that express Ci. Changes in Ci protein levels, detected by an antibody that recognizes an epitope in the carboxy-terminal half of Ci, have been suggested to mediate the positive effects of Hh and the negative effects of PKA on Hh target gene expression in imaginal discs. The effects of PKA on Hh target genes were examined by expressing a mutant regulatory subunit, R*, to reduce PKA activity in embryos. The alterations of wingless, patched and ventral cuticle patterns due to PKA inhibition resemble those induced by low-level ubiquitous expression of Hh but are less pronounced than those elicited by high levels of Hh or strong patched mutations. A constitutively active mouse PKA catalytic subunit transgene (mC*) expressed in Drosophila embryos causes ectopic expression of wingless and patched . Responses to elevated PKA activity require smoothened and cubitus interruptus, but not hedgehog. The absolute requirement for Smo to observe transcriptional induction by PKA hyperactivity is consistent with two mechanisms: (1) either PKA acts on Smo, directly or indirectly, perhaps to uncouple it from the inhibitory influence of Ptc or, (2) alternatively, Smo has Hh-independent activity that acts in parallel with PKA to stimulate wingless and patched expression. There is considerable evidence that phosphorylation can alter activity of G protein-coupled receptors. (Ohlmeyer, 1997).

PKA inhibition, like Hh, leads to increased "carboxy-terminal" Ci staining and Hh target gene expression in embryos. Hh and Smo can stimulate target gene expression at constant Ci levels; increased PKA activity can induce ectopic Hh target gene expression in a manner that requires Smo and Ci activity but does not involve changes in Ci protein concentration. Nevertheless, elevated PKA suppresses the elevation of Ci-C-terminal antibody staining normally elicited by Hh at the borders of each Ci expression stripe. This suggests a branching pathway of Hh signal transduction downstream of Smo and that PKA exerts opposite effects on the two branches. Two PKA targets (direct targeting of Smo and targeting of Ci) with opposing actions on Hh target gene expression can account for the initially surprising observation that both PKA inhibtion and PKA hyperactivity induce wingless and patched expression in embryos. The negative target, relevant to regulating Ci protein levels, is sensitive almost exclusively to reduction of PKA activity. Hh signaling in embryos does not depend on cAMP-dependent regulation of PKA activity (Ohlmeyer, 1997). Direct WG autoregulation (autocrine signaling) is masked by its paracrine role in maintaining hh, which in turn maintains wg. zeste-white3 (shaggy) and patched mutant backgrounds have been used to uncouple genetically this positive-feedback loop and to study autocrine WG signaling. Direct WG autoregulation differs from WG signaling to adjacent cells in the importance of fused, smoothened and cubitus interruptus (ci) relative to zw3 and armadillo (arm). WG autoregulation during this early hh-dependent phase differs from later WG autoregulation due to a lack of gooseberry (gsb) participation (Hooper, 1994).

The simplest possible mechanism by which HH relieves inhibition by Patched takes into consideration the fact that both SMO and PTC are integral membrane proteins and assumes that PTC is inactivated by its association with the HH-SMO complex. It is also possible that PTC in its active form is already associated with SMO in the absence of bound HH. In that case, when HH binds to the SMO-PTC complex, PTC is released from its association with SMO thus abolishing PTC's inhibitory activity on FU. Additionally, PKA becomes inhibited by G protein-coupled signaling (Alcedo, 1996).

Like other members of the serpentine receptor family, Smoothened is also coupled to G proteins. One target of G proteins is Adenylate cyclase, an enzyme that converts Adenosine triphosphospate to cyclic AMP. In turn, cAMP regulates Protein kinase A. Loss of Protein kinase A is known to activate wg in the absence of a HH signal (Jiang, 1995). It is the structural relationship of SMO to G protein coupled serpentine receptors that suggests a coupling of SMO to the regulation of PKA and the subsequent activation of wingless expression (Alcedo, 1996).

smo is required for hedgehog-dependent expression of decapentaplegic. In the case of the wing disc, the principal target of hh is dpp, expression of which is restricted to a thin stripe of cells running along the anterior side of the anterior-posterior compartment boundary. Mosaic imaginal discs, in which small clones of cells within the normal dpp domain lack wild-type smo activity, fail to express dpp in the mutant region. Clones that lack smo and the cAMP-dependent protein kinase (PKA) catalytic subunit, express dpp, indicating that smo is not absolutely required for dpp transcription; rather, it acts upstream of PKA to mediated activation of dpp by hh (van den Heuval, 1996).

Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein

Hedgehog signaling plays a central role in many developmental processes in both vertebrates and invertebrates. The multipass membrane-spanning proteins Patched and Smoothened have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc. Activation of the intracellular signaling pathway occurs when Hh binds to Ptc, an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is thus far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems. To test this model, the Drosophila Smo protein has been overexpressed in vivo and it has been found that increasing the levels of Smo protein per se is not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos reveals distinct patterns of Smo distribution, depending on which region of the protein is detected by the antibody. These findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner (Ingham, 2000).

To analyse the expression of the endogenous Smo protein, an antibody was raised against the membrane-proximal portion of the putative intracellular carboxy-terminal tail of Smo (anti-SmoC antibody) and this was used to stain wild-type and transgenic embryos. In contrast to the ubiquitous distribution of the SMO mRNA, immunohistochemical staining of wild-type embryos with the anti-SmoC antibody reveals a modulated distribution of the protein. Smo accumulates in a series of sequentially repeating stripes, each of which is about one-half a segment in width and spans the parasegmental boundary, the site of Hh activity. Staining of h-Gal4;UAS-smo embryos with the anti-SmoC antibody reveals a significant increase in the levels of Smo protein in the GAL4-expressing segments; strikingly, however, this staining is also restricted to cells flanking the parasegmental boundary. To determine the precise location of these Smo-positive cells, embryos were probed simultaneously with the anti-SmoC antibody and monoclonal antibodies for Engrailed (En), a marker of Hh-expressing cells, and Wg, a marker of Hh-responding cells. Double staining with the anti-En antibody reveals that Smo accumulates in and around cells expressing En; double staining with the anti-Wg antibody shows that Smo also accumulates in cells expressing Wg (Ingham, 2000).

The accumulation of Smo in and around cells secreting Hh protein, strongly suggests that the translation and/or stability of Smo is promoted by Hh activity. To test this possibility, Smo distribution was analyzed in embryos in which Hh is ectopically expressed under the control of the Kr promoter. Such embryos display a ubiquitous expression of Smo between parasegments 5 and 9, precisely the region in which ectopic Hh expression is driven by the Kr-Gal4 driver. Since Hh acts by inhibiting Ptc activity, the effects of Hh on Smo would be expected to be mediated by Ptc. In embryos homozygous for a ptc loss-of-function mutation, the modulated pattern of staining typical of the wild type is lost, indicating that Smo protein accumulates uniformly in the absence of Ptc activity (Ingham, 2000).

The simplest interpretation of these data is that Ptc functions to block the translation or promote the degradation of Smo. And, since the spatial distribution of Smo is unaltered in h-Gal4;UAS-smo transgenic embryos, it would follow that Ptc activity can suppress accumulation of Smo protein independent of the levels at which the gene is transcribed. This effect of Hh/Ptc-mediated signaling on Smo accumulation provides a simple explanation for the lack of an effect of ectopic smo expression, namely that the exogenous protein never accumulates outside the normal domain of Smo activity (Ingham, 2000).

Surprisingly, however, when h-Gal4;UAS-smo embryos were probed with the anti-FLAG antibody, a strikingly different pattern of exogenous protein accumulation is observed. In contrast to the narrow stripes detected by the anti-SmoC antibody, staining is seen throughout each h-Gal4 expression domain. This indicates that the SMO mRNA is translated in all cells in which it is transcribed. It follows that the Ptc-dependent staining pattern revealed by the anti-SmoC antibody reflects a post-translational modification of the Smo protein. One possibility is that Ptc could promote the cleavage of Smo, yielding a relatively stable but functionally inert truncated form of the protein in cells not exposed to Hh. In this connection, it is interesting to note that the SREPB cleavage activating protein (SCAP), with which Ptc shares some homology, acts to promote the cleavage of SREBP by chaperoning the latter from the endoplasmic reticulum to the Golgi. Alternatively, however, it could be that Ptc induces a modification of the Smo protein that masks the epitope recognised by the anti-SmoC antibody; binding of Hh to Ptc would suppress this modification, activating the protein and making it accessible to the antibody. To discriminate between these two possibilities, a second tagged form of Smo was generated, in this case inserting a hemagglutinin (HA) tag at the end of the carboxy-terminal tail. When embryos expressing this construct under h-Gal4 control were stained with an anti-HA antibody, a similar broad distribution of the tagged protein as that revealed by the anti-FLAG antibody is seen. This argues against the cleavage model, but instead suggests that the carboxy-terminal tail undergoes a Ptc-dependent modification. Since the anti-SmoC antibody was raised against an unmodified bacterially expressed protein, it seems most likely that this modification results in a conformational change that exposes epitopes recognised by the antibody. In this regard, it is notable that a putative dominant gain-of-function mutation in the human Smo protein is predicted to change the conformation of the equivalent region of the carboxy-terminal tail against which the anti-SmoC antibody is directed (Ingham, 2000).

Posttranscriptional regulation of Smoothened is part of a self-correcting mechanism in the Hedgehog signaling system

Hedgehog signaling, mediated through its Patched-Smoothened receptor complex, is essential for pattern formation in animal development. Activating mutations within Smoothened have been associated with basal cell carcinoma, suggesting that smoothened is a protooncogene. Thus, regulation of Smoothened levels might be critical for normal development. Smoothened protein levels in Drosophila embryos are regulated posttranscriptionally by a mechanism dependent on Hedgehog signaling but not on its nuclear effector Cubitus interruptus. Hedgehog signaling upregulates Smoothened levels, which are otherwise downregulated by Patched. Demonstrating properties of a self-correcting system, the Hedgehog signaling pathway adjusts the concentrations of Smoothened and Patched to each other and to that of the Hedgehog signal, which ensures that activation of Hedgehog target genes by Smoothened signaling becomes strictly dependent on Hedgehog (Alcedo, 2000).

Posttranscriptional regulation of Smo depends on PKA, an antagonist of the Hh signaling pathway. Thus, PKA activity regulates Smo levels either by stimulating the degradation of Smo or by reducing its rate of synthesis. The mechanism by which Hh regulates the stability of the Smo protein through PKA-dependent proteolysis is favored for two reasons: there is no evidence for PKA-dependent regulation of the rate of synthesis of any protein -- in contrast, it is known that Hh signaling inhibits PKA-dependent proteolytic processing of its nuclear effector Ci. Since several consensus PKA phosphorylation sites are found in the cytoplasmic portions of Smo, PKA might also exert its effect directly on Smo. The phosphorylated form of Smo might be targeted by Slimb to the ubiquitin-ligase complex prior to its proteasome-mediated degradation, a mechanism inhibited by Hh and constitutive Smo signaling. Alternatively, PKA does not act directly on Smo but affects the stability of Smo by activating a protein that destabilizes Smo or by inhibiting a protein that stabilizes Smo (Alcedo, 2000).

A test if Smo levels are uniformly elevated after reducing or completely removing the zygotic Slimb activity in slimb mutant embryos was negative, presumably because of the presence of sufficient wild-type maternal Slimb. After reducing the maternal Slimb activity in hypomorphic slmb1 germline clones, slimb mutant embryos cease to develop by stage 6 and hence can not be tested, since Smo levels are still very low at this stage (Alcedo, 2000).

Why do Hh and Smo signaling upregulate the two Hh-receptor components, Ptc and Smo, at the transcriptional and posttranscriptional level, respectively? What are the advantages of this Hh and Smo signaling system in which Hh inhibits Ptc, which otherwise suppresses Smo signaling and hence downregulates both Smo and Ptc? For convenience, it is assumed in the following a model in which Smo signaling is activated by Hh binding to Ptc as part of a Ptc-Smo receptor complex so far only demonstrated in mammals. Yet none of the considerations presented here are affected by the assumption of such a complex because they are independent of whether Ptc inhibits Smo signaling directly or indirectly. The constitutive activation of the Hh signaling pathway in the absence of Hh is oncogenic. Hence, it is crucial that Smo signaling strictly depends on the presence of Hh and that, in the absence of Hh, constitutive Smo signaling is restricted by Ptc below a threshold necessary for the transcriptional control of Hh target genes. When Hh levels decrease, Smo is destabilized because of the inhibition of Smo signaling by Ptc. The concentration of Smo will be reduced more rapidly than that of Ptc, which continues to be translated from a decreasing concentration of its mRNA, and eventually Smo will reach a reduced steady-state concentration, which is lowest in regions where Hh is absent. When the Ptc concentration falls below a threshold, Smo signaling begins to inhibit its own degradation and to activate transcription of ptc, whose product suppresses Smo signaling and thus again downregulates itself and Smo. Hence, a new steady state is reached at which the levels of Ptc and Smo are reduced to a level corresponding to the low Hh concentration. The sequence of events are expected to be reversed, if the Hh concentration is again increased. Thus, the Hh signaling pathway has the properties of a self-correcting system, since an imbalance between Ptc and Smo or between Hh and the Ptc-Smo receptor is readjusted to equilibrium (Alcedo, 2000).

Although this self-correcting Hh signaling system may appear complex, its properties are probably the simplest solution in ensuring that Smo signaling strictly depends on the concentration of Hh, as apparent from the following considerations. Since Smo signals constitutively in the absence of Ptc, Smo signaling must activate ptc to inhibit its constitutive activity. To avoid an imbalance between the two Hh-receptor moieties, Smo signaling must also upregulate Smo. If Smo levels were independent of Smo signaling, Smo would reach a uniformly high level while the concentration of Ptc would oscillate around an equilibrium since Ptc inhibits Smo signaling on which its synthesis depends. However, in this case Smo would signal even in the absence or at low levels of Hh, which is not what is observed. Therefore, to ensure that Ptc and Smo reach an equilibrium at which Ptc completely inhibits Smo signaling most rapidly in the absence of Hh, Smo regulates its own breakdown (Alcedo, 2000).

The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking

Hedgehog signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, it is shown that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. The mutant PtcSSD protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functions like the wild-type protein in sequestering and internalizing Hh. In addition, PtcSSD preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity (Martin, 2001).

Ptc protein has an SSD, originally identified in HMGCoA reductase and SREBP (sterol regulatory element binding protein) cleavage-activating protein (SCAP), both implicated in cholesterol homeostasis. In addition, it is structurally similar to the Niemann-Pick C1 (NPC1) protein that participates in intracellular cholesterol transport. To address the functional role of the SSD of Ptc, a G-to-A substitution was engineered that causes an Asp583 to change to Asn in the SSD (PtcSSD). This point mutation mimics an Asp-to-Asn mutation in the SSD of SCAP that causes sterol resistance in mutant Chinese hamster ovary cell lines. The Asp mutated in this line is conserved in the SSD of all six SCAP family members known in mouse, human, and C. elegans NPC1 proteins as well as in Ptc. The mutated Drosophila ptc cDNA was introduced into flies under UAS control and overexpressed the mutant protein in the ptc expression domain with ptc-GAL4. The expression of PtcSSD causes embryonic lethality and an almost complete ptc null phenotype. Since the severity of the mutant phenotype correlates directly with the amount of PtcSSD expressed, it has been suggested that PtcSSD competes with the wild-type protein and has a dominant-negative effect (Martin, 2001).

Ptc is located inside the cell, mainly in cytoplasmic vesicles, and when internalization is blocked in shibire mutant embryos, Ptc is associated with the plasma membrane. The large vesicles in which Ptc and Hh accumulate in Hh-responsive cells are endosomes since they colocalized with internalized Texas-red dextran. The same colocalization of Ptc and internalized Texas-red dextran also occurs in ptcS2 mutant clones (which only express the PtcS2 protein. However, Smo protein is not localized with Ptc in the endocytic compartment and is mainly observed along the basolateral plasma membrane. Thus, the Ptc mutation in the SSD alters the subcellular distribution of Ptc, as also occurs upon Hh binding. This preferential location of PtcSSD in punctate structures is apparent when the ectopic expression of PtcSSD is compared with that of PtcWT, which shows a more generalized distribution. Interestingly, there are ptc alleles that do not show this preferential accumulation in endosomes. These alleles induce high Ptc protein levels and do not sequester Hh (Martin, 2001).

Inhibition by steroidal components of both Hh signaling and NPC1-mediated cholesterol transport suggests that Ptc and NPC1 function may have a similar molecular mechanism. A lesion in the NPC1 protein, which is normally found in cytoplasmic vesicles characteristic of late endosomes, produces a general defect in the retrieval and recycling of raft components of the endocytic pathway. Ptc and NPC1 colocalize extensively in vesicular compartments in cotransfected cells. This suggests that the function of both NPC1 and Ptc involves a common vesicular transport pathway (Martin, 2001).

To conclude, the upregulation of Smo protein and the opening of the Hh pathway in PtcSSD mutant cells are related to the preferential accumulation of PtcSSD protein in the endocytic compartment. Therefore, in wild-type cells, subcellular changes in Ptc protein distribution upon Hh binding could impede interaction with Smo and downregulation of Smo protein levels. A future challenge will be to demonstrate that cholesterol modulates the vesicular trafficking of Ptc through its SSD (Martin, 2001).

Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein that binds members of the Hedgehog (Hh) family of signaling proteins and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction. Ptc contains a sterol-sensing domain (SSD), a motif found in proteins implicated in the intracellular trafficking of cholesterol, and/or other cargoes. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein, to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh, and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh, definitive evidence for its function has so far been lacking. The identification and characterization of two missense mutations in the SSD of Drosophila Ptc is described; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. It is speculated that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD (Strutt, 2001).

Conventional models of Hh signaling envisage Ptc to be a ligand binding sub-unit of a Hh receptor that regulates the activity of a signaling subunit, the Smo protein, by inducing conformational changes in the latter. The results of recent studies of Smo in Drosophila have, however, challenged this view and suggested instead that Ptc regulates Smo activity by promoting its posttranslational modification and/or decreasing its stability rather than by locking it into an inactive conformational state. How and where such modifications occur is not known, but the finding that two of three antimorphic alleles of ptc are associated with lesions in the SSD indicates a critical role for this domain in the regulation of Smo. Given that other SSD-containing proteins, such as SCAP and NPC1, are known to mediate trafficking between intracellular compartments, it is tempting to speculate that Ptc may act in a similar manner by directing Smo to an intracellular compartment where it is targeted for modification/degradation. The antimorphic nature of the ptcS2 and ptc34 alleles could be explained if mutation of the SSD abolishes the putative trafficking activity of Ptc without affecting interaction with its cargo. The mutant forms of the protein would thus protect Smo from modification/degradation and leave it free to activate the downstream components of the pathway. By the same token, mutation of the C-terminal tail ( ptc13) might also disrupt the hypothetical trafficking activity; alternatively, it could disrupt cargo interaction. Recent studies have failed to reveal an interaction between the Ptc C-terminal tail and Smo, and this failure favors the former possibility (Strutt, 2001).

Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened

Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Evidence is presented that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity (Denef, 2000).

As a first step toward addressing how Smoothened activity is regulated by Ptc, an antibody to the C-terminal cytoplasmic tail of Smoothened (Smo) protein was produced and Smo expression was examined in imaginal discs. Ptc and Smo are both differentially expressed in A and P cells but in different ways. Ptc is absent from P cells, whereas Smo is expressed at relatively high levels in the P compartment. In the A compartment, Smo levels follow a profile similar to Ptc. Smo protein levels are highest in Hh-responsive anterior cells adjacent to the AP boundary. Smo expression decreases sharply near the boundary and then more gradually across the A compartment. To verify that this accurately reflects Smo protein levels, clones of cells mutant for the smo3 allele were examined. smo3 is associated with a nonsense mutation that truncates the protein after the third transmembrane domain. The C-terminal intracellular portion of the protein recognized by anti-Smo should be absent from the protein encoded by this allele. Clones of mutant cells in both compartments lack Smo antigen. Smo protein is clearly detectable in cells adjacent to the mutant clone in the middle of the A compartment. The level of Smo in wild-type cells adjacent to a more anterior clone is barely distinguishable from background. Although Smo protein expression levels differ in the A and P compartments, SMO mRNA does not appear to be spatially regulated in the wing or leg imaginal discs. In situ hybridization using sense and anti-sense RNA probes did not detect differential expression of SMO mRNA in A and P compartments of the wing disc or of the leg disc. SMO transcript is expressed in a spatially regulated pattern in the brain, which corresponds to the pattern of Smo protein expression (Denef, 2000).

A model for Ptch and Smo activity postulates that Ptc is required for Hh binding, whereas Smo is required to transduce the signal. Ptc blocks the intrinsic signaling activity of Smo, and Hh binding to Ptc alleviates this block and thereby activates Smo. The available evidence suggests that Hh does not bind to Smo in the absence of Ptc nor does Hh activity appear to be required to activate Smo in the absence of Ptc. The prevalent model suggests that Smo might be constitutively active when the inhibitory effects of Ptc are alleviated, and it has been thought that Ptc and Smo exist in a preformed complex at the cell surface that is activated by Hh binding. Three observations from the current analysis support an alternative view of the relationship between Smo and Ptc in Hh signaling: (1) Ptc acts to reduce the level of Smo protein in the absence of Hh. (2) Hh binding triggers removal of Ptc from the cell surface. (3) Hh treatment or removal of Ptc by RNA(i) induces a net increase in phosphorylation of Smo. This correlates with an increase in the level of Smo on the cell surface. These observations raise the possibility that Ptc acts indirectly to regulate Smo activity (Denef, 2000).

How does Hh treatment alter the degree of Smo phosphorylation? Hh binding to Ptc could stimulate the activity of a kinase that phosphorylates Smo. Alternatively, the constitutive activity of Ptc could be mediated by promoting dephosphorylation of Smo. If this is the case, Hh treatment might inactivate a Ptc-dependent phosphatase. The second possibility is favored because dsRNA-mediated depletion of Ptc is sufficient to increase Smo phosphorylation in S2 cells without addition of Hh. In the wing disc, most of the Smo protein comes from the posterior compartment where Ptc is not expressed. Smo from the disc is mostly in the highly phosphorylated form. Thus, in the absence of Ptc, Smo is mainly found in the highly phosphorylated form. The available evidence indicates that Smo is active in the P compartment, but the signal is nonproductive due to the absence of Ci. Taken together, these observations suggest that the highly phosphorylated form of Smo is active in signaling. It is suggested that Ptc activity is mediated by promoting dephosphorylation of Smo and that Hh blocks the ability of Ptc to regulate Smo in A cells near the AP boundary. The relatively low amount of dephosphorylated Smo seen in discs may derive from anterior cells in which Ptc is active because these anterior cells are out of the range of Hh (Denef, 2000).

These findings are consistent with the possibility that Smo is dephosphorylated by a type 2A protein phosphatase. At the concentrations used in these experiments, okadaic acid inhibits PP2A but not PP1-type phosphatases. Smo protein contains consensus sites for phosphorylation by serine/threonine protein kinases, including PKA. The serine/threonine kinase Fused is phosphorylated in response to Hh and plays a role in Hh signaling. In addition, previous work has also implicated PKA and an okadaic acid-sensitive phosphatase in the regulation of Ci phosphorylation. Thus, there appear to be several levels at which phosphorylation and dephosphorylation can regulate Hh signaling activity. The favored model suggests that the constitutive activity of Ptc stimulates activity of a phosphatase that leads to reduced phosphorylation of Smo. Hh binding to Ptc might reduce the ability of Ptc to promote phosphatase activity and allow Smo phosphorylation to increase. According to this view, the state of Smo phosphorylation reflects a balance in the activity of a kinase (which could be constitutively active) and the Ptc-dependent activity of a phosphatase (Denef, 2000).

Evidence has been presented that Hh induces distinct alterations in the subcellular localization of Ptc and Smo proteins. In cells, Hh treatment induces internalization of Ptc and accumulation of Smo at the cell surface. Double labeling studies have shown that Ptc and Hh colocalize in vesicles in the wing disc. In embryos, immunoelectronmicroscopic analysis has shown that Ptc is found in endocytic vesicles and in multivesicular bodies. Multivesicular bodies are intermediates between early and late endosome compartments. The appearance of Ptc in multivesicular bodies is consistent with the possibility that Hh-induced endocytosis targets Ptc to the lysosome for degradation. In this context, it is interesting that Ptc shows sequence similarity to the Niemann-Pick C1 protein, which has been linked to defects in recycling of vesicles in the endocytic and lysosomal pathways. Together, these observations support the view that Hh binding triggers internalization and degradation of Ptc. Under normal circumstances, Hh signaling induces new synthesis of Ptc, which serves to limit the range of Hh movement into the A compartment. These apparently opposing effects on Ptc levels may be reconciled by the idea that this mechanism is used to target Hh for degradation once it has bound Ptc and activated Smo. Clearing Hh from the system may contribute to limiting its range of movement (Denef, 2000).

Smo does not appear to follow Ptc through the endocytic pathway. In contrast, Smo accumulates on the cell surface in Hh-stimulated cells. Accumulation of Smo could reflect Hh-induced transport of Smo from an intracellular pool to the cell surface. Alternatively, Hh-induced phosphorylation might reduce Smo turnover in the membrane, leading to net accumulation. At present, these possibilities cannot be distinguished (Denef, 2000).

Whatever the mechanism for increased accumulation of Smo at the cell surface, these observations suggest that the actively signaling form of Smo is unlikely to be bound by Ptc. If Ptc acts indirectly to regulate Smo activity, the regulatory interaction between Ptc and Smo need not be stoichiometric. It is noted that levels of Smo protein significantly in excess of normal can be rendered functionally inactive by endogenous levels of Ptc. Although it is possible to exceed the capacity of Ptc to regulate Smo activity by overexpression, the results illustrate that Ptc can effectively regulate both Smo activity and Smo protein levels over a considerable range and at levels well above the endogenous level of Smo. These observations support the possibility that Ptc might act indirectly to regulate Smo phosphorylation, with concomitant effects on subcellular localization, stability, and activity (Denef, 2000).

Altered localization of Smoothened protein activates Hedgehog signal transduction

Hedgehog (Hh) signaling is critical for many developmental events and must be restrained to prevent cancer. A transmembrane protein, Smoothened (Smo), is necessary to transcriptionally activate Hh target genes. Smo activity is blocked by the Hh transmembrane receptor Patched (Ptc). The reception of a Hh signal overcomes Ptc inhibition of Smo, activating transcription of target genes. Using Drosophila salivary gland cells in vivo and in vitro as a new assay for Hh signal transduction, the regulation of Hh-triggered Smo stabilization and relocalization was investigated. Hh causes Smo (GFP-Smo) to move from internal membranes to the cell surface. Relocalization is protein synthesis-independent and occurs within 30 min of Hh treatment. Ptc and the kinesin-related protein Costal2 (Cos2) cause internalization of Smo, a process that is dependent on both actin and microtubules. Disruption of endocytosis by dominant negative dynamin or Rab5 prevents Smo internalization. Fly versions of Smo mutants associated with human tumors are constitutively present at the cell surface. Forced localization of Smo at the plasma membrane activates Hh target gene transcription. Conversely, trapping of activated Smo mutants in the ER prevents Hh target gene activation. Control of Smo localization appears to be a crucial step in Hh signaling in Drosophila (Zhu, 2003).

The salivary gland experiments show that Smo is normally present in a meshwork of organelles in the cytoplasm. Upon reception of a Hh signal, Smo protein moves quickly to the surface. This change in subcellular localization could be due to release from a tether and movement, or to a change in the net flow of protein cycling through membrane compartments. If Smo, for example, normally circulates to and from the surface, Ptc could facilitate the inward movement. When Hh binds to Ptc and inactivates it, Smo would cycle to the surface and remain there. This idea is consistent with the increased surface Smo observed when endocytosis is blocked with the shibire or Rab5 mutation (Zhu, 2003).

Surface Smo localization correlates fully with Hh target gene transcription but the amount of Smo protein does not. The apparent increase in the amount of Smo at the cell surface that occurs when Hh signal is received may be caused by sequestration of Smo away from proteases in internal membrane compartments. Smo moves to the surface on addition of Hh or removal of Ptc, conditions that activate target gene transcription (Zhu, 2003).

The mutant forms of fly Smo designed to mimic human oncogenic SMO alleles are also located at the surface. The basis for the tumors is hypothesized to be inappropriate activation of Hh (perhaps Shh) target genes in the skin and cerebellum, targets that are insufficiently restrained by Ptc1 or other regulators. The oncogenic forms of Smo appear to be resistant to inhibition by Ptc and at least one of them is clearly resistant to teratogenic drugs that bind Smo and inhibit responses to Hh signaling. These experiments suggest that the fly versions of the oncogenic proteins are also at least partially resistant to Ptc and are refractory to the Ptc-imposed internalization of Smo protein. Overexpression of the oncogenic mutants causes dramatic changes in wing patterning and anterior outgrowth. Conversely, tagging oncogenic Smo mutants with an ER retrieval motif, KKDE at the C terminus prevents Hh signaling. This addition of only three amino acids (one K is already present at the C terminus of fly Smo) drastically changes the activities and localization of the protein (Zhu, 2003).

Forms of Smo with surface localization signals added at the C terminus are in fact enriched at the cell surface and these proteins activate Hh target gene transcription. These results suggest the importance of subcellular localization for regulating Smo activity. Smo normally resides in internal compartments of unknown character, and moves in response to Hh and other regulators. It is unknown whether surface localization per se is important, or instead a movement to new compartments that happen to be located at or near the cell periphery (Zhu, 2003).

Smo protein is predicted to have seven transmembrane domains. The C terminus of Smo protein, where GAP43 and GPI linkage sequences were added, is predicted by most modeling programs to be cytoplasmic, but given the absence of any data on the actual transmembrane topology of Smo, two tags with different properties were used. The GAP43 tether works from the cytosolic side, whereas the GPI tether is believed to work only if the tagged part of the protein is on the luminal side of the membrane (Zhu, 2003).

The successful activation of Smo by the GAP43 tether is consistent with the presumed cytoplasmic topology of the C terminus. This topology would mean, however, that the GPI tether is not formed because the necessary machinery to cleave the GPI signal is located in the lumen of the ER. The appearance of the surface-localized Smo differs for the two tags, with the GPI-motif protein giving a continuous pattern that connects adjacent cells and the GAP43 sequence giving discernably separate layers of protein on two adjacent cells. Two possible explanations of the GPI results are that the tag alters the Smo protein topology so that the protein becomes GPI-linked or that the tag changes Smo conformation to make an activated molecule. It has been proposed that Smo cycles between two different conformations. The movement of Smo to a new location might shift the balance toward the active state of Smo, for example if the pH, ionic milieu, lipid composition, or presence of other proteins in the new compartment alter Smo conformation. The GPI signal could alter the Smo conformation to an active form, and in this case surface localization could be a consequence, rather than cause, of activation (Zhu, 2003).

Flies lacking smo function are unable to activate target genes in response to Hh signaling, in contrast to ptc mutants, which activate target genes inappropriately even in the absence of Hh signal. Double mutants that lack both smo and ptc function fail to activate Hh target gene transcription, indicating that the failure of repression by Ptc is irrelevant if Smo is not present to allow activation. On this basis, Ptc has been viewed as an opponent of Smo function (Zhu, 2003).

Smo is similar to the Frizzled (Fz) Wnt receptors in primary sequence and presumed structure, but Smo has no known ligand and as yet has not been found to bind a Wnt protein. Surface localization could be critical if it allows an as-yet-unknown activating ligand to bind Smo. A limiting amount of ligand might explain why over-producing Smo alone does not have a strong effect on development (Zhu, 2003).

The regulation of Smo by Ptc remains a mystery. Hh could inactivate Ptc by binding to it either on the surface or in internal vesicles. If Ptc cycles between surface and interior regions of the cell, the binding by Hh could change Ptc so that it is less likely to travel to the surface. In contrast to Hh inactivation of Ptc, mutational inactivation of Ptc does not lead to an internal location, at least in the case of the dominant-negative form that accumulates at the surface. Ptc, possibly through a transporter function, could change organelle contents or composition so that Smo changes conformation to its active form. Alternatively Ptc could either detach Smo from an intracellular tether or alter the movements of vesicles bearing Smo protein. Smo, previously cycling to and from the surface, would accumulate on the surface and increase in amount (Zhu, 2003).

Signaling from Smo, through a Galpha protein or other means, could alter the cytoplasmic Ci complex and change Ci processing to control target gene transcription. Experiments in frog melanophores and zebrafish embryos have suggested possible Galpha(i) involvement in Hh signal transduction. Since both studies involved overexpression of proteins, it is unclear as yet whether Smo acts through G protein signal transduction. An alternative, more direct interaction with the cytoplasmic protein complex seems possible, for example, if Smo controls encounters between the complex and the as-yet-unknown protease that cleaves Ci (Zhu, 2003).

The dominant-negative form of Ptc is present at the cell surface and could compete for an association between Ptc and Smo, compete for an association between Ptc and another protein, or associate with wild-type Ptc and inactivate its activity. The distinct locations of Ptc and Smo in fly imaginal disc cells and in salivary gland cells, suggest that the bulk of each protein is not in association with the other protein. There could nonetheless be some of the proteins in association, below the level of detection by staining techniques. Although little or no Ptc-Smo association was seen by immunoprecipitation from cultured cells, transient associations would not be seen, particularly if the arrival of Ptc or Hh/Ptc in a Smo-containing organelle immediately caused the departure of Smo (Zhu, 2003 and references therein).

Ptc contains a sequence related to 'sterol-sensing domains (SSDs)' that have been implicated in altered functions or stability of proteins involved in lipid metabolism. Mutations in the Ptc SSD reduce the ability of Ptc to repress Smo function. It is possible that Ptc regulates Hh signaling through effects on membrane trafficking. Analysis of mice with mutations in the open brain (opb) gene lend further support for the potential involvement of protein trafficking in Hh signal regulation. opb encodes Rab23, which negatively regulates Hh signal transduction. Rab GTPases coordinate the budding, fission, transport, docking, and fusion of vesicles as they move from one cellular location to a target compartment. The shuttling of Smo and Ptc between internal membrane compartments and the cell surface presumably requires Rab activity. Disruption of endocytosis by dominant-negative Shibire and by Rab5 manipulation prevents both Smo and Ptc internalization (Zhu, 2003).

Movement of Smo to the surface requires actin and tubulin components of the cytoskeleton, though the relevant motors are unknown. Cos2 is an unusual member of the kinesin family, with sequence features at odds with conventional ATPase binding site structure. Cos2 could be either a motor or a tether. Cos2 could have a role in controlling movements of vesicles that contain Smo. Overproduction of Cos2 alters GFP-Smo localization, and furthermore, prevents Hh from bringing much GFP-Smo to the surface, and the GFP-Smo that does reach the surface is located in discreet dots. Ptc also blocked Hh from bringing GFP-Smo to the surface, but no such dots were observed. Overexpression of a presumably irrelevant other motor protein, Nod, has no effect on localization of GFP-Smo. Cos2 production may therefore specifically cause the movement of Smo-containing organelles to discreet locations on the membrane, either tethering them to the cytoskeleton at specific locations or causing a coalescence effect at random locations. Cos2 has been envisioned as functioning as part of a cytoplasmic complex whose activity in processing the Ci transcription factor is controlled by Smo. The present data suggest a new function in which the complex (oralternatively, Cos2 independently of the complex), feeds back to alter Smo activity. It is interesting that both GFP-Smo (when Cos2 and Hh were coexpressed) and PtcDN-YFP exhibits a similar punctate cell surface localization pattern. PtcDN may function through competing with endogenous Ptc, raising an intriguing alternative possibility that Cos2 may interact directly with Smo to control Smo subcellular localization (Zhu, 2003).

Human oncogenesis by activated Smo and the importance of the Hh pathway in numerous developmental events in all animals makes understanding Hh signal transduction critical. The present approach has identified new interactions between components of the pathway. The causal link between surface location and activity during Hh signaling, with Ptc inactivation, with Smo oncogenic mutants and with mislocalization of Smo add strong evidence that the localization of Smo is a critical regulatory step in Hh signaling (Zhu, 2003).

Inhibition of Hh signaling by direct binding of cyclopamine to Smoothened

Plants of the genus Veratrum have a long history of use in the folk remedies of many cultures, and members of the jervine family of alkaloids, constituting a majority of Veratrum secondary metabolites, have been used for the treatment of hypertension and cardiac disease. The association of Veratrum californicum with an epidemic of sheep congenital deformities during the 1950s raised the possibility that jervine alkaloids are also potent teratogens. Extensive investigations by the U.S. Department of Agriculture subsequently confirmed that jervine and cyclopamine (11-deoxojervine) given during gestation can directly induce cephalic defects in lambs, including cyclopia in the most severe cases. It is now known that the teratogenic effects of jervine and cyclopamine are due to their specific inhibition of vertebrate cellular responses to the Hedgehog (Hh) family of secreted growth factors, as first suggested by similarities between the Vertarum-induced developmental malformations and holoprosencephaly-like abnormalities associated with loss of Sonic hedgehog (Shh) function. In accordance with this general mechanism, cyclopamine also has shown some promise in the treatment of medulloblastoma tumors caused by inappropriate Hh pathway activation (Chen, 2002 and references therein).

Using photoaffinity and fluorescent derivatives, it has now been shown that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo (Chen, 2002).

Since both cyclopamine and Ptch negatively regulate Smo activity, how Ptch activity influences the ability of Smo to bind cyclopamine was investigated. Increased levels of mouse Ptch expression in COS-1 cells dramatically enhances the photoaffinity cross-linking of post-ER Smo by 125I-labeled photoaffinity reagent-tagged cyclopamine (PA-cyclopamine). In contrast, the labeling of ER-localized Smo was not affected, and cellular concentrations of either Smo form were not altered by Ptch expression. Treatment of the Smo- and Ptch-expressing cells with the N-terminal domain of Shh (ShhN) is able to reverse the effect of Ptch expression on PA-cyclopamine/Smo cross-linking, confirming its dependence on Ptch activity (Chen, 2002).

These results provide some insights into the regulation of Smo by Ptch. (1) Ptch appears to act only on post-ER Smo, since the PA-cyclopamine cross-linking of ER-localized Smo is independent of Ptch expression levels. This subcellular compartmentalization of Ptch action is consistent with previous observations that Ptch is primarily localized to endosomal/lysosomal vesicles and the plasma membrane. (2) The ability of Ptch expression to significantly increase post-ER Smo labeling by PA-cyclopamine without influencing overall protein levels suggests that the effect of Ptch activity alters Smo conformation and that Ptch and cyclopamine promote inactive Smo states that may be structurally related (Chen, 2002).

How Ptch influences Smo conformation remains enigmatic, despite extensive genetic analyses of the Hh pathway. Although it was initially proposed that Ptch and Smo form a heteromeric receptor, it is now believed that Smo activity is modulated by Ptch in an indirect, nonstoichiometric manner (Taipale, 2002). In the case of the Frizzled family of seven-TM receptors, which are closely related to Smo in structure, receptor activation involves the binding of Wnt ligands to the Frizzled CRD and recruitment of an LDL receptor-related protein. No analogous protein interactions have been associated with Smo activation, and removal of the Smo CRD does not appear to significantly alter Smo function or its suppression by Ptch (Chen, 2002 and references therein).

These observations coupled with the susceptibility of Smo to cyclopamine suggest that Smo regulation may involve endogenous small molecules rather than direct protein-protein interactions. Consistent with this model, Ptch is structurally related to the resistance-nodulation-cell division family of prokaryotic permeases and to the Niemann-Pick C1 protein, which are both capable of transporting hydrophobic molecules. Ptch action might similarly affect the subcellular and/or intramembrane distribution of endogenous molecules, thus influencing Smo activity by altering the localization of a Smo ligand. Alternatively, this Ptch activity could influence membrane structure and Smo trafficking; a shift in Smo localization might then be accompanied by activity-modulating changes in the molecular composition of specific subcellular compartments (Chen, 2002 and references therein).

The demonstration of cyclopamine binding to Smo establishes the mechanism of action for this plant-derived teratogen. These studies show that cyclopamine interacts with the Smo heptahelical bundle, thereby promoting a protein conformation that is structurally similar to that induced by Ptch activity. Equally important, these studies reveal the molecular basis for cyclopamine's antitumor activity and validate Smo as a therapeutic target in the treatment of Hh-related diseases. Aberrant Hh pathway activation has been associated with several cancers, such as medulloblastoma and basal cell carcinoma, and many of these tumors involve mutations in Ptch or Smo. As a specific Smo antagonist, cyclopamine may be generally useful in the treatment of such cancers, a therapeutic strategy further supported by the absence of observable toxicity in cyclopamine-treated animals. Additional Smo antagonists might also be discovered through small molecule screens for specific Hh pathway inhibitors, thus comprising a class of pharmacological agents with possible utility in the treatment of Hh-related oncogenesis (Chen, 2002).

Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail

The Hedgehog (Hh) family of secreted proteins controls many aspects of growth and patterning in animal development. The seven-transmembrane protein Smoothened (Smo) transduces the Hh signal in both vertebrates and invertebrates; however, the mechanism of its action remains unknown. Smo lacking its C-terminal tail (C-tail) is inactive, whereas membrane-tethered Smo C-tail has constitutive albeit low levels of Hh signaling activity. Smo is shown to physically interact with Costal2 (Cos2) and Fused (Fu) through its C-tail. Deletion of the Cos2/Fu-binding domain from Smo abolishes its signaling activity. Moreover, overexpressing Cos2 mutants that fail to bind Fu and Ci but retain Smo-binding activity blocks Hh signaling. Taken together, these results suggest that Smo transduces the Hh signal by physically interacting with the Cos2/Fu protein complex (Jia, 2003).

The most surprising finding of this study is that the Smo C-tail suffices to induce Hh pathway activation. Overexpressing the membrane-tethered Smo C-tail (Myr-SmoCT, Sev-SmoCT) blocks Ci processing, induces dpp-lacZ expression, and stimulates nuclear translocation of Ci155. Myr-SmoCT is refractory to Ptc inhibition and activates Hh-pathway independent of endogenous Smo. Membrane tethering appears to be crucial for the Smo C-tail to activate the Hh pathway; untethered SmoCT has no signaling activity. This is consistent with observations that cell surface accumulation of Smo correlates with its activity (Jia, 2003).

Although the Smo C-tail has constitutive Hh signaling activity, it does not possess all the activities associated with full-length Smo. For example, overexpressing Myr-SmoCT in A-compartment cells away from the A/P compartment boundary does not significantly activate ptc and en, which are normally induced by high levels of Hh. In addition, Myr-SmoCT cannot substitute endogenous Smo at the A/P compartment boundary to transduce high levels of Hh signaling activity, since boundary smo mutant cells expressing Myr-SmoCT fail to express ptc in response to Hh (Jia, 2003).

The failure of the Smo C-tail to transduce high Hh signaling activity is due to its inability to antagonize Su(fu). Although Myr-SmoCT blocks Ci processing to generate Ci75, the activity of Ci155 accumulated in Myr-SmoCT-expressing cells is still blocked by Su(fu); removal of Su(fu) function from Myr-SmoCT-expressing cells allows Ci155 to activate ptc to high levels. Because Myr-SmoCT stimulates nuclear translocation of Ci155, the inhibition of Ci155 by Su(fu) in Myr-SmoCT-expressing cells must rely on a mechanism that is independent of impeding Ci nuclear translocation (Jia, 2003).

Several observations prompted a determination of whether Smo can transduce the Hh signal by physically interacting with the Cos2/Fu complex: (1) although Smo is related to G-protein-coupled receptors, no genetic or pharmacological evidence has been obtained to support the involvement of a G-protein in a physiological Hh signaling process; (2) Myr-SmoCT can interfere with the ability of endogenous Smo to transduce high levels of Hh signaling activity, which can be offset by increasing the amount of full-length Smo. This implies that Myr-SmoCT may compete with full-length Smo for binding to limiting amounts of downstream signaling components. (3) Extensive genetic screens failed to identify Hh signaling components that may link Smo to the Cos2/Fu complex (Jia, 2003).

Using a coimmunoprecipitation assay, it was demonstrated that Smo interacts with the Cos2/Fu complex both in S2 cells and in wing imaginal discs, and the Smo C-tail appears to be both necessary and sufficient to mediate this interaction. The Cos2/Fu-binding domain was narrowed down to the C-terminal half of the Smo C-tail (between amino acids 818 and 1035). Furthermore, both the microtubule-binding domain (amino acids 1-389) and the C-terminal tail (amino acids 990-1201) of Cos2 interact with Smo. Since none of these Cos2 domains binds Fu, this implies that the Cos2/Smo interaction is not mediated through Fu. Ci is also dispensable for Smo/Cos2/Fu interaction; Smo binds Cos2/Fu in S2 cells in which Ci is not expressed. However, the results did not rule out the possibility that Smo could interact with the Cos2/Fu complex through multiple contacts. For example, Smo could simultaneously contact Cos2 and Fu. Nor was it demonstrated that binding of Cos2 to Smo is direct. Indeed, no protein-protein interaction between Smo and Cos2 was detected in yeast. It is possible that a bridging molecule(s) is required to link Smo to the Cos2/Fu complex. Alternatively, Smo needs to be modified in vivo in order to bind Cos2. It has been shown that Hh stimulates phosphorylation of Smo; hence, it is possible that phosphorylation of Smo might be essential for recruiting the Cos2/Fu complex (Jia, 2003).

Several lines of evidence suggest that Smo/Cos2/Fu interaction is important for Hh signal transduction. (1) Deletion of the Cos2-binding domain from Smo, either in the context of full-length Smo or the Smo C-tail, abolishes Smo signaling activity. (2) Overexpressing Cos2 deletion mutants that no longer bind Fu and Ci but retain a Smo-binding domain intercept Hh signal transduction. Genetic evidence has been provided that Cos2 has a positive role in transducing Hh signal in addition to its negative influence on the Hh pathway, since Ci155 is no longer stimulated into labile and hyperactivity forms by high levels of Hh in cos2 mutant cells. In light of the finding that Smo interacts with Cos2/Fu, the simplest interpretation for a positive role of Cos2 is that it recruits Fu to Smo and allows Fu to be activated by Smo in response to Hh (Jia, 2003).

Of note, interaction between SmoCT and Cos2/Fu per se is not sufficient for triggering Hh pathway activation. For example, Myr-SmoCTDelta625-818, which binds Cos2/Fu to the same extent as Myr-SmoCT, does not possess Hh signaling activity. The fact that Myr-SmoDeltaCT625-730 and Myr-Smo730-1035 can activate the Hh pathway suggests that Smo sequence between amino acids 730 and 818 is essential. This domain may recruit factors other than Cos2/Fu to achieve Hh pathway activation. Alternatively, it might target SmoCT to an appropriate signaling environment (Jia, 2003).

An important property of Hh family members in development is that they can elicit distinct biological responses via different concentrations. How different thresholds of Hh signal are transduced by Smo to generate distinct transcriptional outputs is not understood. The results suggest that Smo can function as a molecular sensor that converts quantitatively different Hh signals into qualitatively distinct outputs. In the absence of Hh, the cell surface levels of Smo are low. In addition, the Smo C-tail may adopt a 'closed' conformation that prevents it from binding to Cos2/Fu. Low levels of Hh partially inhibit Ptc, leading to an increase of Smo on the cell surface. In addition, the Smo C-tail may adopt an 'open' conformation, which allows Smo to bind the Cos2/Fu complex and inhibit its Ci-processing activity. Low levels of Hh signaling activity can be mimicked by overexpression of either full-length Smo or membrane-tethered forms of the Smo C-tail. High levels of Hh completely inhibit Ptc, resulting in a further increase in Smo signaling activity. Hyperactive Smo stimulates the phosphorylation and activity of bound Fu, which in turn antagonizes Su(fu) to activate Ci155. Consistent with this, Fu bound to Myc-Smo was found to became phosphorylated in response to ectopic Hh (Jia, 2003).

The Smo sequence N terminus to SmoCT (SmoN) appears to be essential for conferring high Smo activities. It is not clear how SmoN modulates the activity of SmoCT. SmoN might recruit additional effector(s) or target SmoCT to a microdomain with a more favorable signaling environment. Alternatively, SmoN might function as a dimerization domain that facilitates interaction between two SmoCTs, as in the case of receptor tyrosine kinases. It is also not clear how Smo/Cos2/Fu interaction inhibits Ci processing. One possibility is that Smo/Cos2 interaction may cause disassembly of the Cos2/Ci complex, which could prevent Ci from being hyperphosphorylated; Cos2/Ci complex formation might be essential for targeting Ci to its kinases. Consistent with this view, Ci is barely detectable in the Cos2/Fu complex bound to Smo (Jia, 2003).

Physical association of the receptor complex with a downstream signaling component has also been demonstrated for the canonic Wnt pathway whereby the Wnt coreceptor LRP-5 interacts with Axin, a molecular scaffold in the Wnt pathway. Hence, Hh and Wnt/Wg pathways appear to use a similar mechanism to transmit signal downstream of their receptor complexes (Jia, 2003).

Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2

The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Evidence is presented that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo (Lum, 2003).

Early studies of Cos2 suggested primarily a negative role for Cos2 in pathway regulation, as manifested by phenotypic analysis of cos2 mutations and by a requirement for Cos2 in cytoplasmic retention of Ci and in its proteolytic processing to produce CiR. More recent studies have suggested a potential positive role based on a requirement for Cos2 in transcriptional activation of gene targets associated with highest levels of pathway activity. This study extends the evidence for such a positive role by demonstrating: (1) a requirement for Cos2 in mediating a series of Hh-induced biochemical changes in pathway components; (2) an association between the Cos2/Fu/Ci complex and Smo; (3) a direct interaction between Smo and Cos2, and (4) a requirement for Cos2 in highest level Hh pathway response in cultured cell reporter assays and Hh-induced morphogenesis in the dorsal cuticle (Lum, 2003).

Based on the sequence relationship between Smo and GPCRs, previous speculation and experimental work has focused on the possibility that Smo may interact with heterotrimeric G proteins. G protein components have been systematically targeted using RNAi in a cultured cell signaling assay, and no significant role has been found for G proteins in transcriptional regulation via Ci. A potential role cannot be ruled out for G proteins or other mediators in cellular responses to Hh signaling that do not involve transcriptional regulation via Ci/Gli. For example, a recently described chemoattractant activity for Shh in axon guidance appears to be mediated by Smo, yet proceeds in a short time scale and with a local cell polarity that suggests a possible nonnuclear mechanism of response (Lum, 2003).

In searching for other mediators of information transfer from membrane to cytoplasm, it was surprising to find cytoplasmic components copurifying with Smo. Since these were the only Drosophila proteins identified in the bands excised, it is concluded that these complexes were highly pure, and that Smo associates stably with components of the cytoplasmic complex in vivo. It is further demonstrated that Smo interacts directly with Cos2, which scaffolds this complex. Consistent with these findings, articles presenting genetic evidence for a role of the Smo cytoplasmic tail in Hh signaling and evidence suggesting a physical interaction between Smo and Cos2 were published during final review of this work. Additional direct association of Smo with other complex components have not been ruled out (Lum, 2003 and references therein).

The identification of a complex that includes both Ci and Smo immediately suggested that recruitment of the cytoplasmic complex to Smo upon Hh stimulation might be critical for pathway activity. Cos2 plays a central role in mediating this association, functioning both as a scaffold that brings together cytoplasmic components (the Cos2 complex) and as a sensor that monitors the state of pathway activation by interacting with Smo at the membrane (Lum, 2003).

In the unstimulated state, Smo levels are low, and most of the Cos2 complex therefore is not associated with or influenced by Smo; even the small fraction of Cos2 complex associated with Smo may be negative, since Smo may not be in an active state. The negative form of the Cos2 complex presumably mediates production of CiR and prevents nuclear accumulation of Ci, resulting in a net suppression of transcriptional targets. In the intermediate state, present after a few minutes of stimulation, Smo protein has become activated by Hh stimulation but has not yet accumulated. Therefore, despite a positive state for the specific Cos2 complexes affected by Smo, the low level of activated Smo protein is insufficient for interaction with most of the Cos2 complexes. The net outcome with regard to transcriptional targets thus remains negative (Lum, 2003).

In stimulated cells, activated Smo exerts a pervasive influence on the Cos2 complex, either through a stable association or through a transient association with enduring effects. This association presumably involves a direct binding interaction between a portion of the Smo cytotail and the Cos2 cargo and stalk domains. The evidence suggests that both activation and accumulation of Smo are critical, as evidenced by the observation that moderate Smo overexpression alone is unable to fully activate the pathway in the absence of Hh stimulation. Similarly, a cycloheximide block of Smo accumulation dramatically limits the biochemical changes normally induced by Hh stimulation, and cotransfection experiments demonstrate that transition of Cos2 from a pathway suppressor to activator requires adequate levels of activated Smo. The activation of transcriptional targets resulting from pathway stimulation presumably result from the positive action of Cos2 on Fu and from loss of the ability to produce CiR (Lum, 2003).

It is the dual action of Cos2 in promoting formation of CiR and in stabilization and possible activation of Fu that leads to the apparent dual negative and positive roles of Cos2 in pathway regulation. Pathway activation induced by loss of Cos2 thus results from loss of CiR, but this activation is only partial because Fu is also destabilized, and the pathway-suppressing action of Su(fu) is unchecked. Consistent with this interpretation, a combined loss of Cos2 and Su(fu) results in maximal pathway activation irrespective of the presence of Hh (Lum, 2003).

How does Cos2 function? Motor proteins recently have been found to play a role in regulation of transmembrane receptors, as in the case of rhodopsin and mannose-6-phosphate receptor. These motor proteins apparently regulate receptor localization but do not play a direct role in receptor function. A recent report suggests that Cos2 overexpression indeed may influence Smo localization. The evidence, however, suggests that Cos2 also functions as a primary sensor of the state of pathway activation by interacting with Smo at the membrane and by scaffolding and stabilizing downstream pathway components (Lum, 2003).

Kinesins likely share an evolutionary origin with G proteins and myosins, and all three types of proteins use a conserved mechanism to couple nucleotide hydrolysis to dramatic conformational changes in protein structure. Kinesins utilize this mechanism to generate force that allows movement on microtubules. Sequences essential for microtubule binding, for nucleotide binding, and for motor function in other kinesins, however, are not conserved among Cos2 sequences in the Diptera and are not required for Cos2 function in cultured cells. It is still possible, however, that Cos2 retains the capacity for a conformational shift that may be triggered by Smo activation. If so, then this conformational change may involve the Cos2 cargo domain, which binds to Smo at the cytoplasmic tail and is likely required for Smo responsiveness (Lum, 2003).

Whatever its mechanism of activation, evidence points to a role for the atypical kinesin Cos2 as a scaffold and sensor that functions as the pivotal component in transduction of pathway activation from the seven transmembrane receptor Smo to the latent cytoplasmic transcription factor Ci. In addition to stabilizing Fu and mediating forward signaling events that affect other cytoplasmic components and Ci, Cos2 is also required for the accumulation of activated Smo, a critical aspect of producing a full response to Hh signal. Cos2 thus functions not just as a passive sensor for the state of pathway activation at the membrane, but is also an active participant in the cellular dynamics of transition from the unstimulated to the stimulated state. These activities are all the more remarkable in view of the well-recognized role played by Cos2 in maintaining the unstimulated state of the Hh pathway, and together these findings suggest that Cos2 dynamics are a critical determinant of intracellular Hh pathway response and regulation (Lum, 2003).

Hrs mediates downregulation of Smoothened and other signalling receptors in Drosophila

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).

Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homolog, Vps27p, also have a UIM and bind to ubiquitin. The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Jékely, 2003 and references therein).

To determine whether Hrs is generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the Drosophila ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation at the cell cortex could also be observed. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).

An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27. Genetic studies in mice and Drosophila (Lloyd, 2002) have also shown that cells mutant for hrs have enlarged endosomes, possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes, and can also be used to specifically label endosomes. The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells is compared, an enlargement of FYVE-positive structures is observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).

Hrs affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs that were analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures were also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).

To test whether the requirement for Hrs was limited to RTKs, other types of signalling receptors were analysed. The Hedgehog receptor Patched and the Hedgehog signal transducer Smoothened are multi- and seven-pass transmembrane proteins, respectively. Thickveins (Tkv) is a type-I serine-threonine kinase receptor for the TGF-ß family ligand Dpp. Notch is a single-pass transmembrane protein that undergoes specific proteolytic cleavage upon activation. Interestingly, hrs mutant follicle cells show a marked accumulation of each of these receptors. As for RTKs, most of the receptor molecules accumulate intracellularly and show significant colocalization with the ubiquitinated protein signal. Thus, Hrs has a general role in regulating the sorting and degradation of diverse classes of signalling receptors. The homotypic adhesion molecule Shotgun is not affected visibly in hrs mutant cells. The latter observation is in agreement with observations that nonsignalling transmembrane proteins are not upregulated in hrs mutant animals (Lloyd, 2002). Either the trafficking of these proteins is independent of Hrs function or they have a low turnover rate in the examined tissues (Jékely, 2003).

The high degree of overlap between the signal for each of the receptors and the signal for ubiquitinated proteins means that the receptors accumulate in roughly the same endosomal compartment. This, together with the increase in receptor levels in hrs mutant cells, suggests that these receptors are degraded through the same Hrs-dependent pathway. Ubiquitination of the inhibitory Smad7 by the E3 ubiquitin ligase Smurf2 has been shown to target the Smad7-TGF-ß receptor complex for lysosomal degradation. In follicle cells, a similar complex may be sorted for degradation in an Hrs-dependent manner. It has been argued that the turnover of Hedgehog receptors is strongly regulated and may be critical for signalling, but a role of ubiquitination in this event has not been reported. The observation that Patched and Smoothened accumulate in compartments highly enriched in ubiquitinated proteins in hrs mutant cells suggests that trafficking of Patched and Smoothened is also regulated by ubiquitination (Jékely, 2003).

When analysing hrs mutant clones, an increase of ubiquitinated proteins at the cell cortex was occasionally noticed in addition to the intracellular accumulation. Some cortical accumulation could also be observed directly for the signalling receptors, in particular for Tkv. This accumulation could be due to inefficient endocytosis from the plasma membrane or increased recycling of endocytosed proteins. Hrs does not appear to be required directly for endocytosis (Lloyd, 2002), but downstream defects may 'clog up' the endocytosis machinery. Hrs can also affect receptor recycling. Overexpression of Hrs in tissue culture cells increases the retention of ubiquitinated transferrin receptors. The strong intracellular accumulation of receptors in hrs mutant cells could therefore either be due to defective sorting towards lysosomal degradation or due to defective post-endocytic retention, a concomitant general increase in the steady-state levels of the receptors at the plasma membrane, and therefore in endosomes. The first explanation is favored because increased surface levels of receptors or ubiquitinated proteins were often not detected even when strong intracellular accumulation was evident. Receptors therefore seem to be retained intracellularly, rather than recycled, in hrs mutant cells. Hrs is most likely not the only factor responsible for the post-endocytic retention of receptors. Redundancy in sorting to the vacuole has been reported for the yeast alpha-factor receptor Ste3p. In this case, Vps27p and Hse1 have overlapping roles to sort Ste3p to the vacuolar lumen (Jékely, 2003).

The bulk of Hrs-dependent downregulation of signalling receptors appears to be constitutive as well. Hedgehog acts very early in egg chamber development, and patched-lacZ, which reflects Hedgehog activity, has very restricted expression. Downregulation of Patched in the stage 10 egg chamber should therefore be ligand independent. Smoothened protein is, in turn, controlled by Patched. EGFR ligands are highly enriched and active at the dorsal side of the egg chamber, whereas a PVR ligand is present throughout the oocyte. However, for both receptors, the level of receptor accumulation in hrs mutant cells is similar throughout the follicular epithelium. Signal-induced endocytosis is well established for acute stimulation of signalling receptors, in particular RTKs. Yet signalling does not appear to control the bulk of receptor turnover in follicle cells. The physiological levels of stimulatory ligands may be relatively low compared to the levels used for acute stimulation experiments (Jékely, 2003).

Precise regulation of signalling strength is essential for interpreting morphogen gradients and thus for correct patterning during development. The control of signalling receptor levels at the cell membrane is an important aspect of this regulation. It is therefore of interest to know how receptor levels are regulated under physiological conditions. The results presented here indicate that diverse classes of signalling receptors undergo constitutive (ligand-independent) ubiquitination, endocytosis and Hrs-dependent degradation. The efficiency of this traffic affects the responsiveness of cells to patterning signals: blockage of trafficking in hrs mutants can sensitize cells to a low level of signalling molecules, whether RTK ligands (Lloyd, 2002) or Dpp (this study). However, it does not lead to ligand-independent signalling, supporting the conclusion that most endocytosed receptor molecules are not activated. Ligand-induced endocytosis may also occur, but affects only a minority of receptor molecules in this in vivo context. Constitutive turnover of receptors may serve as quality control by removing damaged receptors or receptors in partially formed signalling complexes. A constant flux of all receptor molecules may also facilitate the efficient clearance of activated receptors (Jékely, 2003).

Identification of a functional interaction between the Smoothened and Costal2

Hedgehog signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a microtubule-associated Hedgehog signaling complex (HSC). At a minimum, the HSC consists of the Kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). In response to HSC activation, the ratio between repressor and activator forms of Ci is altered, determining the expression levels of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. A functional interaction is described between Smo and Cos2 that is necessary for Hh signaling. It is proposed that this interaction is direct and allows for activation of Ci in response to Hh. This work fills in the last major gap in understanding of the Hh signal transduction pathway by suggesting that no intermediate signal is required to connect Smo to the HSC (Ogden, 2003).

To determine whether Cos2 and Smo could interact directly, a directed yeast two-hybrid assay was used. The cytoplasmic carboxyl-terminal domain of Smo (SmoC) was used in the two-hybrid assay, since the signaling capabilities of Smo appear to reside within this domain. The carboxyl-terminal domain of Smo interacts with Cos2, though this interaction appears less efficient than that of Cos2 with Fu. This interaction is specific and reproducible, since there is no growth when the open reading frame of Cos2 is inserted in the reverse orientation. These results demonstrate that the carboxyl-terminal domain of Smo is sufficient to associate with Cos2 and that this association appears to be direct. Combined with immunoprecipitation and immunofluorescence data, the yeast two-hybrid results provide strong evidence that Smo and Cos2 directly associate and that the association occurs within the intracellular signaling portion of Smo (Ogden, 2003).

To determine whether Hh signaling would affect the Cos2-Smo interaction, Smo was immunoprecipitated from S2 cell lysates prepared from cells transfected with Hh expression or control vectors. Cos2 and Fu coimmunoprecipitate with Smo at similar levels regardless of Hh activation status. Phosphorylation-induced mobility shifts of Cos2 occurs in Hh-transfected cells, verifying that Hh signaling is intact. The modest increase observed in Cos2 immunoprecipitating with Smo in response to Hh stimulation may be accounted for by Smo protein stabilization in response to Hh. These results suggest that interactions between Smo, Cos2, and Fu are relatively stable and independent of Hh activation status (Ogden, 2003).

To verify that Hh activation does not modify Smo-Cos2 association in vivo, Smo immunoprecipitations were performed from embryos engineered to overexpress Ptc, Hh, or neither. Embryos overexpressing Ptc serve as a source of cells in which Hh signaling is inactive due to repression of Smo by Ptc, while embryos overexpressing Hh serve as a source of Hh-activated cells. Mobility shifts of Cos2, Fu, and Smo, which have previously been attributed to Hh-induced phosphorylation, confirm that Hh or Ptc have turned Hh signaling on or off in these embryos. An equal amount of Cos2 was observed coimmunoprecipitating with Smo from wild-type, Ptc, and Hh embryo lysates. In two separate experiments, it was estimated that 3% of Cos2, 4% of Fu, and 3%-8% of Smo were recovered in coimmunoprecipitates. By contrast, 50% of Fu was recovered by Cos2 immunoprecipitation, while negligible amounts of Fu, Cos2, or Smo were recovered in Fz immunoprecipitates. These results demonstrate that a small percentage of total Cos2 and Smo are associated in a high-affinity association, and the percentage associated does not change due to Hh signaling (Ogden, 2003).

Expression of a chimera of SmoC fused to a myristate membrane-targeting sequence (Myr-SmoC) induces phenotypes in Drosophila similar to cos2 loss-of-function mutations; weak Hh responses are activated, while strong Hh responses are inhibited. Myr-SmoC drives all Hh responses to a weak activation state in Drosophila and requires endogenous Smo to do so. Although the mechanism by which Myr-SmoC acts is unknown, dosage dependence of the effect suggests that it interferes with signaling by competing with endogenous Smo for Cos2. A similar epitope-tagged construct was expressed in cultured cells to test the hypothesis that Myr-SmoC interferes with signaling by binding to Cos2. Using a Myc epitope tag to specifically immunoprecipitate Myr-SmoC, it was found that both Cos2 and Fu associate with Myr-SmoC. These data support the directed two-hybrid experiment, showing that the carboxyl-terminal domain of Smo is sufficient to interact with Cos2. Further, Myr-SmoC functions was found to be a potent inhibitor of Hh signaling, able to inhibit Hh-dependent transcription in a dose-dependent fashion. These results indicate that even in the absence of Hh, Ci activity is effectively reduced by Myr-SmoC. Thus, Myr-SmoC does not constitutively activate Ci in this reporter assay. It is proposed that Myr-SmoC can act as a dominant negative by binding endogenous Cos2. This argument is bolstered by genetic evidence showing that increasing Cos2 levels in vivo can suppress the overgrowth phenotype associated with expressing Myr-SmoC in flies. These results are consistent with the hypothesis that association between Smo and Cos2 is necessary for Hh signaling to be propagated to its ultimate effector, the transcription factor Ci (Ogden, 2003).

Two scenarios are proposed that may account for the observation that Smo and Cos2 association is not altered in response to Hh. The first possibility is that Smo and Cos2 may be held in an associated but inactive state in the absence of Hh stimulation, presumably through the function of Ptc. Hh stimulation would relieve Ptc-mediated repression of the Smo-Cos2 complex to allow Smo relocalization to the plasma membrane. The Kinesin-like properties of Cos2 and its direct interaction with Smo may facilitate this relocalization. A second possibility is that the dynamics of association are changed in response to Hh, such that Smo and Cos2 association turns over more rapidly in the process of creating the active form of Ci (Ogden, 2003).

Divergence of hedgehog signal transduction mechanism between Drosophila and mammals

The Hedgehog (Hh) signaling pathway has conserved roles in development of species ranging from Drosophila to humans. Responses to Hh are mediated by the transcription factor Cubitus interruptus (Ci; GLIs 1-3 in mammals), and constitutive activation of Hh target gene expression has been linked to several types of human cancer. In Drosophila, the kinesin-like protein Costal2 (Cos2), which associates directly with the Hh receptor component Smoothened (Smo), is essential for suppression of the transcriptional activity of Ci in the absence of ligand. Another protein, Suppressor of Fused [Su(Fu)], exerts a weak negative influence on Ci activity. Based on analysis of functional and sequence conservation of Cos2 orthologs, Su(Fu), Smo, and Ci/GLI proteins, Drosophila and mammalian Hh signaling mechanisms have been found to diverge; in mouse cells, major Cos2-like activities are absent and the inhibition of the Hh pathway in the absence of ligand critically depends on Su(Fu) (Varjosalo, 2006).

The evidence indicates that a significant divergence in the mechanism of Shh signal transduction has occurred between vertebrates and invertebrates at the level of Smo, Cos2, and Su(Fu). The results indicate that major Cos2-like activities are absent in mouse cells based on four observations: (1) domains in Smo that are required in Drosophila to bind to Cos2 are not required for mSmo function; (2) mouse Shh signaling is insensitive to expression of Drosophila Cos2, but can be rendered Cos2 sensitive by replacing the mSmo C-terminal domain with the dSmo C-terminal domain; (3) expression of the Smo C-terminal domain which, in Drosophila, inactivates Cos2 has no effect in the mouse in vivo or in vitro; (4) overexpression or RNAi-mediated suppression of mouse Cos2 homologs has no effect on Hh signaling, even under sensitized conditions. These results are also consistent with divergence of the sequence of domains involved in Cos2 binding in Ci/GLI proteins and Smo between insects and mammals (Varjosalo, 2006).

Although the RNAi experiments targeting Cos2 orthologs Kif7 and Kif27 were performed under conditions in which negative regulators of GLI2 were limiting, they could be argued to be consistent with a model in which multiple kinesins with Cos2-like activity would act in a redundant fashion in mammals. By loss-of-function studies of individual kinesins in cell culture or in mice it would be difficult to obtain conclusive evidence against such a model due to the potential redundancy of multiple members of the kinesin family. However, several other in vitro and in vivo experiments that were presented directly contradict such a model. These include RNAi analyses targeting multiple Kif proteins, the analysis of loss of function of mSmo domains, and the lack of effect of overexpression of myristoylated-mSmoC and the Cos2 orthologs Kif7 and Kif27. In addition, no kinesin with Cos2-like activity could be found by extending the analyses to several other kinesins, which show homology to Cos2 but have different fly orthologs (Varjosalo, 2006).

In contrast to the case in Drosophila, Su(Fu) has a critical role in suppression of the mammalian Hh pathway in the absence of ligand, and loss of Su(Fu) function results in dramatic induction of GLI transcriptional activity. The results are also consistent with the studies that show that loss of Su(Fu) in mouse embryos results in complete activation of the Hh pathway, in a fashion similar to the loss of Ptc. These results are particularly surprising in light of the central role of Cos2 and a minor role of Su(Fu) in Drosophila. Together, these results also clearly show that mouse cells and embryos lack a Cos2-like activity that, in Drosophila, can completely suppress the Hh pathway in the absence of Su(Fu). However, the results should not be taken as evidence against novel proteins (including kinesins not orthologous to Cos2) acting in mammalian cells between Smo and GLI proteins with mechanisms that are distinct from those used by Drosophila Cos2. Several reports have, in fact, described such vertebrate-specific regulators of Hh signaling, including SIL, Iguana, Rab23, Kif3a, IFT88, IFT172, MIM/BEG4, and β-arrestin2 (Varjosalo, 2006).

The results also shed light on some known differences in the function of the Hh pathway in Drosophila and mammals. Mutations and small molecules affecting conformation of Smo transmembrane domains have a strong effect in mammals, but they have little effect in Drosophila. Interestingly, the Smo transmembrane domain is required for regulation of Su(Fu) activity, whereas the Smo C-terminal domain is critical for inhibition of Cos2 activity. Thus, based on the data, manipulations that affect the Smo transmembrane domain would be predicted to affect Su(Fu) and therefore to have a limited role in Drosophila and a major effect in mammals (Varjosalo, 2006).

Although there are differences in mouse and Drosophila Smo functional domains, and a lack of conservation of Smo phosphorylation sites, conservation of Smo function at a level not involving Cos2 is supported by the observation that mutation of a conserved isoleucine (I573A in mSmo) results in loss of both mouse and Drosophila Smo activity, yet does not result in a loss of Cos2 binding to dSmo. In addition, dSmo proteins that are activated by phosphomimetic mutations are constitutively stabilized; yet, they are partially responsive to Hh, suggesting that, in addition to stabilization and phosphorylation, other, potentially conserved mechanisms could be required to generate fully active Smo in Drosophila as well (Varjosalo, 2006).

In the mSmo C terminus, six residues between amino acids 570 and 580 were identified that resulted in significant loss of mSmo activity. The predicted secondary structure for this region is an α helix, in which these residues would reside on the same side, raising the possibility that, together with the third Smo intracellular loop, this region may form an interaction surface involved in inactivation of Su(Fu) or activation of Ci/GLI (Varjosalo, 2006).

Recent results have indicated that Su(Fu) acts as a tumor suppressor in medulloblastoma, and it has been suggested that medulloblastomas associated with loss of Su(Fu) result, in part, from activation of the Wnt pathway. However, consistent with the lack of a Wnt phenotype of Su(Fu) mutations in Drosophila, in the current experiments, a Wnt pathway-specific reporter is not activated by shRNAs targeting Su(Fu). Given observations that Su(Fu) is critically important in the suppression of the mammalian Hh pathway in the absence of ligand, and the fact that Hh pathway activation is required for growth of a form of medulloblastoma induced by mutations in Patched, it is likely that constitutive activation of the Hh pathway is also essential for growth of medulloblastomas associated with the loss of Su(Fu) (Varjosalo, 2006).

In a wider context, the results demonstrate that signal transduction mechanisms of even the major signaling pathways are not immutable, but that they can be subject to evolutionary change. The divergence may have occurred after the separation of the vertebrate and invertebrate lineages. However, some evidence also suggests that functional divergence may have occurred much later in evolution. Although mutants of Fused or Cos2 orthologs of zebrafish have not been identified, zebrafish homologs of Fused and Cos2 act in the Hh pathway based on morpholino antisense injections. In contrast to these findings, mice deficient in mouse ortholog of Drosophila Fused do not have a Hh-related phenotype, and mouse orthologs of Cos2 do not affect Hh signaling. Hh-related phenotypes can be observed in zebrafish by morpholino-mediated targeting of other genes as well, such as β-arrestin2, whose loss in mice does not result in a Hh-related phenotype. It is widely appreciated that multiple types of embryonic insults result in Hh-like phenotypes, such as holoprosencephaly. Thus, it is possible that the zebrafish phenotypes observed are caused by the morpholino injection process itself. Alternatively, there may also be significant differences between the mechanism of Hh signaling between vertebrate species (Varjosalo, 2006).

Because of the strong conservation of Su(Fu) in both invertebrate and vertebrate phyla, the presence of a Cos2 binding domain only in insect Smo, and the divergence of the Cos2 proteins from the kinesin family, the simplest explanation of the data is that Su(Fu) represents the primordial Ci/GLI repressor, and that the Cos2-like functionality has evolved specifically in the invertebrate lineage. The results, thus, also raise the possibility that multicomponent pathways evolve, in part, by insertion of novel proteins between existing pathway components. This mechanism potentially explains a challenging aspect of evolutionary biology regarding the emergence of signaling pathways with multiple specific components (Varjosalo, 2006).

The contributions of protein kinase A and smoothened phosphorylation to hedgehog signal transduction in Drosophila

Protein kinase A (PKA) silences the Hedgehog (Hh) pathway in Drosophila in the absence of ligand by phosphorylating the pathway's transcriptional effector, Cubitus interruptus (Ci). Smoothened (Smo) is essential for Hh signal transduction but loses activity if three specific PKA sites or adjacent PKA-primed casein kinase 1 (CK1) sites are replaced by alanine residues. Conversely, Smo becomes constitutively active if acidic residues replace those phosphorylation sites. These observations suggest an essential positive role for PKA in responding to Hh. However, direct manipulation of PKA activity has not provided strong evidence for positive effects of PKA, with the notable exception of a robust induction of Hh target genes by PKA hyperactivity in embryos. This study shows that the latter response is mediated principally by regulatory elements other than Ci binding sites and not by altered Smo phosphorylation. Also, the failure of PKA hyperactivity to induce Hh target genes strongly through Smo phosphorylation cannot be attributed to the coincident phosphorylation of PKA sites on Ci. Finally, it has been shown that Smo containing acidic residues at PKA and CK1 sites (see Double-time) can be stimulated further by Hh and acts through Hh pathways that both stabilize Ci-155 and use Fused kinase activity to increase the specific activity of Ci-155 (Zhou, 2006; full text of article).

When the role of PKA in Hh signaling was first discovered it appeared that PKA acted simply to silence the pathway in the absence of Hh. This aspect of PKA function has been studied further, revealing that it is conserved in vertebrate Hh signaling and can be explained adequately by the phosphorylation of three clustered consensus PKA sites on Ci-155. Loss of these sites, loss of PKA activity, and even the consequences of excessive PKA activity in wing discs all lead to a coherent picture of how PKA silences Ci and the Hh signaling pathway in the absence of Hh. This role of PKA had disguised recognition of any potential positive role for PKA in transduction of an Hh signal on the basis of simply manipulating PKA activity. Indeed, a positive role for PKA in Hh signaling was clearly revealed only by altering PKA (and PKA-primed CK1) phosphorylation sites in Smo; changes to alanine residues eliminated activity and changes to acidic residues endowed some constitutive activity. A number of significant questions remain. Are the consensus PKA sites on Smo actually phosphorylated by PKA and only by PKA, and is phosphorylation of Smo by PKA required to transmit an Hh signal? Does Smo with acidic residues at PKA and CK1 sites mimic the consequences of phosphorylation at those sites, and does it elicit the normal process of Hh pathway activation (Zhou, 2006 and references therein)?

Smo absolutely requires PKA sites for activity. Furthermore, those sites can be phosphorylated by PKA in vitro to prime phosphorylation of adjacent CK1 sites, and those CK1 sites are also essential for Smo activity. Hence, Smo PKA sites must be critical in their phosphorylated form and elimination of the relevant protein kinase activity should prevent all responses to Hh. Expression of a dominant-negative PKA regulatory subunit (R*) in embryos does substantially reduce Fu phosphorylation induced by endogenous or ectopically expressed Hh, consistent with the idea that PKA is the major protein kinase that phosphorylates Smo on PKA sites in embryos. However, PKA inhibition with R* in embryos does not prevent all Hh-stimulated phosphorylation of Fu or Hh-dependent maintenance of wg expression. Since PKA inhibition by R* is likely incomplete it is not possible to distinguish whether these residual responses to Hh result from phosphorylation of Smo by residual PKA activity or by another protein kinase, but it should be noted that PKA inhibition by R* is sufficient to produce very high levels of Ci-155, indicative of a complete block in Ci-155 processing (Zhou, 2006).

In wing discs PKA-C1 activity can be eliminated cleanly in large clones using null alleles. PKA-C1 (formerly named DC0) is the major PKA catalytic subunit in flies and the only PKA catalytic subunit with demonstrated developmental functions, even though at least one other gene encodes an equivalent biochemical activity. Loss of PKA-C1 activity in wing disc clones does reduce Hh signaling, as revealed most clearly by strongly reduced or absent expression of En at the AP border. This deficit of PKA-C1 mutant clones at the AP border can be complemented by expressing SmoD1-3 in place of wild-type Smo. This supports the idea that PKA-C1 must phosphorylate Smo for Hh to elicit maximal pathway activity, which is required for strong induction of En. It is not so straightforward to determine whether Hh requires PKA-C1 activity to induce target genes such as collier (col) or ptc, which require lower levels of Hh pathway activity. This is because loss of PKA-C1 by itself induces strong ectopic ptc and col expression. Nevertheless, when induction of col in PKA-C1 mutant clones was largely suppressed by reducing the dose of ci, it was clear that Hh still induced high levels of col in PKA-C1 mutant clones at the AP border and that this induction required Smo activity. Thus, Smo retains some but not maximal activity in response to Hh when PKA-C1 activity is lost, implying that another kinase can phosphorylate Smo at PKA sites in wing discs. This inference is also supported by the observations that Smo is stabilized in anterior cells when its PKA sites are substituted by alanine residues but not when PKA-C1 activity is eliminated (Zhou, 2006).

In contrast to the limited effects of eliminating PKA-C1 activity on Smo activity and protein levels, the same manipulations of PKA-C1 completely block processing of Ci-155 to Ci-75 and strongly activate Ci-155 in wing discs. Why might Smo and Ci-155 show different sensitivities to PKA-C1? One possibility is that scaffolding molecules may allow special access of PKA-C1 to Ci-155 that is not available to other kinases that might otherwise phosphorylate PKA sites. Indeed, Cos2 does appear to ensure efficient phosphorylation of Ci-155 by PKA-C1 by binding to both components. However, Cos2 also binds to Smo and therefore presumably also provides similarly enhanced access for PKA-C1. A more likely explanation of the different responses of Smo and Ci-155 to PKA-C1 manipulation concerns the stoichiometry of phosphorylation. A key functional consequence of Ci-155 phosphorylation is the binding of Slimb, and this requires extensive phosphorylation of Ci-155 primed by each of the three relevant PKA sites. Thus, any significant reduction in the rate of phosphorylation of these sites might be translated into strong stabilization of Ci-155. Conversely, since Smo retains considerable activity in the absence of PKA-C1 it is speculated that a low rate of phosphorylation of Smo at PKA sites may suffice for it to be active (Zhou, 2006).

The discovery that substitution of multiple PKA and CK1 site Serines of Smo with acidic residues conferred constitutive activity provoked the simple hypothesis that activation of Smo by Hh can be attributed largely to an Hh-stimulated increase in phosphorylation at these sites. Investigations of the properties of Smo with acidic residues at PKA and CK1 sites (SmoD1-3) and of the consequences of forced phosphorylation of Smo do not support this simple hypothesis (Zhou, 2006).

It was found that Hh can increase pathway activity in cells expressing SmoD1-3. This effect is small in wing discs, where (overexpressed) SmoD1-3 has strong constitutive activity. However, in embryos SmoD1-3 exhibited no clear constitutive activity but transduced a normal response to Hh. Thus, Hh must elicit changes in Smo activity other than phosphorylation at PKA and CK1 sites that are sufficiently important to convert pathway activity from a silent state to being fully active in embryos. It is speculated that these (unknown) changes are conserved elements of all Hh signaling pathways and that phosphorylation of Drosophila Smo at PKA and CK1 sites, which are not conserved in vertebrate Smo proteins, is a prerequisite for Drosophila Smo to undergo these Hh-dependent changes (Zhou, 2006).

It was also found that excess PKA activity and CK1 activity cannot reproduce the ectopic activation of Hh target genes induced by expression of SmoD1-3. This was true despite attempts to sensitize Hh target gene induction by eliminating Su(fu) or by providing additional processing-resistant Ci-155. An analogous difference in the potency of SmoD1-3 and excess PKA and CK1 activity was observed when using Fu phosphorylation as a measure of Hh pathway activity in wing discs (Zhou, 2006).

Why are excess PKA and CK1 activities not sufficient to activate Smo? One possibility is that overexpression of PKA or CK1 did not effectively stimulate Smo phosphorylation. This explanation is not favored because both of the protein kinases used are thought to associate with Cos2 and therefore should have good access to Smo, and analogous overexpression studies show that each can lower Ci-155 levels at the AP border, implying that they induce significant changes in Ci-155 phosphorylation (Zhou, 2006).

Another possibility is that PKA or CK1 may have targets other than Smo that reduce Hh signaling pathway activity, obscuring the effects of any potential activation mediated by Smo phosphorylation. Ci-155 is certainly one such target but this confounding influence was excluded by coexpression of a Ci mutant lacking all known regulatory PKA sites and also by measuring Fu phosphorylation in addition to Hh target gene activation. It is conceivable that there are additional inhibitory targets for PKA in the Hh pathway because it was observed that the induction of ptc-lacZ in posterior wing disc cells by a PKA-resistant Ci variant (Ci-H5m) was, surprisingly, reduced by excess PKA activity (Zhou, 2006).

Finally, the favored explanation is that Smo with acidic residues at PKA and CK1 sites behaves significantly differently from Smo that is phosphorylated at those sites. It has been argued that phosphorylation is essential for the activity of Smo in the presence of Hh but also targets Smo for degradation in the absence of Hh. It is further speculated that Hh might normally stabilize the phosphorylated state of Smo rather than actively promoting Smo phosphorylation and that acidic residues might mimic Smo activation by phosphorylation without simultaneously promoting Smo degradation in the absence of Hh. In this scenario SmoD1-3 would accumulate and exhibit constitutive activity, especially when overexpressed, but it would not be possible to accumulate activated Smo very effectively in the absence of Hh by increasing only its rate of phosphorylation at PKA and CK1 sites. The hypothesis that Hh stabilizes phosphorylated Smo rather than promoting Smo phosphorylation is also consistent with the earlier conjecture that Smo activation by Hh requires only a low rate of phosphorylation at PKA sites (Zhou, 2006).

A significant question for the future is how phosphorylation of Smo contributes to its activity. Some clues have been made available from examining the properties of SmoD1-3 in wing discs. SmoD1-3 stabilizes Ci-155, induces phosphorylation of Fu, shows substantial dependence on Fu kinase activity for induction of Hh target genes and can suffice for strong induction of anterior En expression in wing discs. These results suggest that SmoD1-3 activates two genetically separable aspects of Hh signaling (Ci-155 stabilization and the Fu kinase signaling pathway) that are sometimes hypothesized to correspond to two biochemically distinct pathways. The nonphysiological circumstances of using high levels of expression and acidic residues in place of phosphorylation may contribute to one or the other of the apparent dual attributes of SmoD1-3 in Hh signaling. Nevertheless, it appears that phosphorylation of Smo at PKA and CK1 sites at least makes Smo competent to activate each known aspect of the Hh signaling pathway. This fits with the idea that Smo phosphorylation may be constitutive but necessary to make Smo competent to respond to Hh (Zhou, 2006).

It was found that strong ectopic activation of the Hh target genes, wg and ptc, by excess PKA activity in embryos is the consequence of two distinguishable responses. First, PKA does appear to induce target genes through Ci binding sites, consistent with enhancing Smo activity through phosphorylation. However, this response alone would result in only a very small induction of Hh target genes. The salient evidence is that PKA hyperactivity induces (1) detectable, but very limited, ectopic expression of a reporter gene that essentially contains only Ci binding sites, (2) clear ectopic expression of a wg reporter gene that depends on the presence of Ci binding sites, and (3) a small increase in Fu phosphorylation. Second, PKA hyperactivity induces wg and ptc transcription principally through regulatory elements other than Ci binding sites and through a mechanism that does not require a change in phosphorylation at Smo PKA sites. The salient evidence is that the response to excess PKA is greatly enhanced if regulatory elements from the wg and ptc genes other than just Ci binding sites are present and that wg and ptc are strongly induced by excess PKA activity even when the only Smo protein present has acidic residue substituents at PKA and CK1 sites (Zhou, 2006).

The dual consequences of excess PKA described above clarify a potential misconception in the literature that PKA can strongly activate the Hh pathway through Smo and substantiate the idea that excess PKA produces only a small activation of the Hh pathway through phosphorylation of Smo, whether assayed in wing discs or embryos. These results also raise the question of the nature and physiological significance of the pathway that connects excess PKA activity to induction of wg and ptc through enhancer elements other than Ci binding sites (Zhou, 2006).

PKA is known to phosphorylate many proteins that can influence transcription and thus its ability to activate wg and ptc through sites other than Ci binding sites when hyperactive may simply be an artifact of this nonphysiological condition An alternative possibility is that this consequence of excess PKA activity exposes a regulatory mechanism that is relevant to target gene activation by Hh in embryos. There is some evidence for transcription factors other than Ci contributing to induction of Hh target genes in embryos. Furthermore, it is clear that there must be interactions between Ci and other gene-specific transcription factors that underlie both the different sensitivity of genes with equivalent Ci binding sites to activation by Ci-155 and repression by Ci-75 and the tissue-specific responses of most genes to Hh. Whether Hh signaling affects the activity or interactions of transcription factors that collaborate with Ci is not presently known (Zhou, 2006).

An intriguing aspect of the ectopic induction of wg and ptc by excess PKA through sites other than Ci binding sites is its dependence on concomitant activation through Ci binding sites. Thus, induction of wg and ptc by excess PKA requires both Smo and Ci activities and requires functional Ci binding sites within the Deltawg-lacZ reporter gene. Even the PKA sites on Smo are required for wg to respond to excess PKA, consistent with the idea that some activation of Smo is required. There is as yet no indication that Hh signaling normally involves the PKA-responsive regions of wg and ptc enhancers that can collaborate with Ci binding sites. Indeed, both Ci-Grh-lacZ and FE-lacZ reporters, which lack key regulatory regions required for a strong response to excess PKA activity, are clearly induced by Hh. There are, however, caveats to this evidence; induction of Ci-Grh-lacZ depends on the synthetic Grh binding sites as well as its Ci binding sites and the FE-lacZ reporter is induced only poorly by Hh in comparison to the ptc-lacZ reporter that includes PKA-responsive elements. Thus, it remains possible that the Hh signal is transmitted largely through Ci and supplemented by contributions from enhancer elements other than Ci binding sites, including those that are responsive to PKA. One pathway that is known to supplement Hh-induced wg expression in embryos is the Wg autoregulation pathway. However, this does not appear to be relevant to the PKA-responsive elements under discussion here because PKA hyperactivity did not substitute for the requirement for Wg activity to maintain stripes of wg expression and PKA hyperactivity also induces ectopic ptc expression, which does not depend on Wg activity for its expression. In the future, the clearest way to test the significance for Hh signaling of regulatory elements responsive to excess PKA will be to define and then alter those regulatory elements (Zhou, 2006).

Phosphorylation of the atypical kinesin Costal2 by the kinase Fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling

The Hedgehog (Hh) family of secreted proteins is involved both in developmental and tumorigenic processes. Although many members of this important pathway are known, the mechanism of Hh signal transduction is still poorly understood. In this study, the regulation of the kinesin-like protein Costal2 (Cos2) by Hh was analyzed. A residue on Cos2, serine 572 (Ser572), is necessary for normal transduction of the Hh signal from the transmembrane protein Smoothened (Smo) to the transcriptional mediator Cubitus interruptus (Ci). This residue is located in the serine/threonine kinase Fused (Fu)-binding domain and is phosphorylated as a consequence of Fu activation. Although Ser572 does not overlap with known Smo- or Ci-binding domains, the expression of a Cos2 variant mimicking constitutive phosphorylation and the use of a specific antibody to phosphorylated Ser572 showed a reduction in the association of phosphorylated Cos2 with Smo and Ci, both in vitro and in vivo. Moreover, Cos2 proteins with an Ala or Asp substitution of Ser572 were impaired in their regulation of Ci activity. It is proposed that, after activation of Smo, the Fu kinase induces a conformational change in Cos2 that allows the disassembly of the Smo-Fu-Cos2-Ci complex and consequent activation of Hh target genes. This study provides new insight into the mechanistic regulation of the protein complex that mediates Hh signalling and a unique antibody tool for directly monitoring Hh receptor activity in all activated cells (Reul, 2007).

These data show that phosphorylation of Cos2 residue Ser572 is necessary for the full activation of Hh signalling, and that this phosphorylation is dependent on the kinase Fu. It is likely that Fu directly phosphorylates Cos2 on Ser572, but it was not possible to purify an activated Fu kinase to confirm this. The phosphorylation of this residue strongly decreased the association of Cos2 with both Ci and Smo, an important step in the regulation of the cytoplasmic anchoring of Ci. By contrast, Cos2-572A, a Cos2 mutant that cannot be phosphorylated at Ser 572, remained associated with Smo and Ci but was much less sensitive to Hh regulation; this is because both its restraining activity on Ci and its association with Ci were only minimally sensitive to the presence of Fu and to the activation of Hh signalling. Phosphorylation of Ser572 of Cos2 induces the partial disassembly of the protein complex (Reul, 2007).

The data show that Cos2 phosphorylated on Ser572 does not bind Smo. However, previous studies have shown that Cos2 is phosphorylated and is pulled down by Smo in response to Hh stimulation. How can these data be reconciled? First, it is possible that not all Cos2 proteins that bind to Smo are phosphorylated. Indeed, only a limited fraction of Cos2 and Fu are sensitive to Hh activation. This is clearly observed with Fu (only 50% of the protein undergoes an electromobility shift upon Hh activation), but is more difficult to quantify with Cos2 because of its very small and diffused electromobility shift. Nevertheless, if Cos2 behaves similarly to Fu, it would mean that 50% of the total Cos2 (corresponding to the non-modified protein in Hh-treated cells) should be able to bring enough Smo down to be detectable in immunoprecipitates. Second, it is possible that Smo still binds to phosphorylated Cos2 on Ser572, but with much less affinity. Third, phosphorylation on Ser572 is not responsible for all Cos2 mobility shift, because Cos2-572A still shifts upon OA treatment, suggesting that other phosphorylated sites are present. Therefore, some phosphorylated isoforms that are not phosphorylated on Ser572 might also be associated with Smo. It is thus possible that this study has revealed only one of a series of sequential phosphorylation events on Cos2 that ultimately lead to the complete dissociation of Cos2 from Smo. Finally, it is worth mentioning that more Smo is present in the Cos2 IP from Hh-treated cells than in non-Hh treated cells. This is thought to simply reflect an increased level of Smo resulting from Hh signalling activation, and not the Hh-dependent regulation of the efficiency of the interaction of Smo with Cos2 (Reul, 2007).

The role of the Cos2 protein in the complex is to serve as a platform to allow both positive and negative regulators to be brought into close proximity with Smo and Ci. Thus, the role of Cos2 in transmitting a response can be masked by the role of Cos2 in limiting pathway activity in the absence of Hh. At low concentrations, it is able to stimulate Hh reporter activity in vitro and engrailed expression in vivo. But in Cos2-572A-expressing cells, engrailed expression was lower than in wild-type discs, and the in vitro stimulation of Hh signalling could not be potentiated by Fu activity. Moreover, the restraining activity of Cos2-572A on Ci could not be counteracted by Hh or Fu in vitro. Therefore, it is proposed that the Ser572 to Ala substitution on Cos2 rendered Cos2 less sensitive to Hh and Fu regulation. Because Cos2-572A still binds to its partners, it could bring Fu into proximity with its other targets. Indeed, it is likely that Fu activation leads not only to the direct phosphorylation of Cos2 but also to direct changes in Ci and/or other partners, such as Sufu. This explains why Cos2-572A is still able to stimulate Hh signalling, albeit not to its highest level (Reul, 2007).

From the Cos2-572A results, one could wonder why Cos2-572D did not constitutively activate the pathway. Because the Cos2-572D form is in a 'frozen' state compared with the wild-type form, cycles of phosphorylation/dephosphorylation are blocked and thus Cos-572D cannot participate in the Hh complex signalling anymore. The data show that constitutively phosphorylated Cos2 and endogenous phospho-Cos2 are bound to Fu but are dissociated from Smo and Ci. Therefore, Fu bound to phosphorylated Cos2 would be absent from the complex, preventing the release of all the cytoplasmic anchors from Ci (Reul, 2007).

Because the Cos2 Ser572 residue is not part of the Ci- or Smo-binding domains, but phosphorylation of this site nevertheless leads to the dissociation of these two proteins from Cos2, it is proposed that the Fu-mediated modification of Cos2 induces the protein to undergo a conformational change that leads to the disassembly of the complex. The disassembly is partial because phosphorylated Cos2 and Fu are still associated. Interestingly, it has been proposed that the binding of Cos2, Sufu and Fu to Ci masks a nuclear localisation site on Ci (Ci-NLS). A conformational change that supports this idea: that disassembly of the complex is necessary to expose the Ci-NLS and for consequent nuclear translocation (Reul, 2007).

Hedgehog regulates smoothened activity by inducing a conformational switch

Hedgehog (HH) morphogen is essential for metazoan development. The seven-transmembrane protein smoothened (SMO) transduces the HH signal across the plasma membrane, but how SMO is activated remains poorly understood. In Drosophila, HH induces phosphorylation at multiple Ser/Thr residues in the SMO carboxy-terminal cytoplasmic tail, leading to its cell surface accumulation and activation. This study provides evidence that phosphorylation activates SMO by inducing a conformational switch. This occurs by antagonizing multiple Arg clusters in the SMO cytoplasmic tail. The Arg clusters inhibit SMO by blocking its cell surface expression and keeping it in an inactive conformation that is maintained by intramolecular electrostatic interactions. HH-induced phosphorylation disrupts the interaction, and induces a conformational switch and dimerization of SMO cytoplasmic tails, which is essential for pathway activation. Increasing the number of mutations in the Arg clusters progressively activates SMO. Hence, by employing multiple Arg clusters as inhibitory elements counteracted by differential phosphorylation, SMO acts as a rheostat to translate graded HH signals into distinct responses (Zhao, 2007; full text of article).

The prevalent view regarding SMO regulation is that SMO is activated as a result of subcellular compartmentation. This study provides substantial evidence that SMO activity is also regulated by a conformational switch. In particular, an autoinhibitory domain (SAID) was identified in the Drosophila SMO cytoplasmic tail, containing multiple Arg clusters that keep SMO in a closed inactive conformation through intracellular electrostatic interaction. HH-induced phosphorylation disrupts such interaction and triggers a conformational switch and increased proximity of SMO cytoplasmic tails, which may further promote recruitment and interaction of intracellular signalling complexes. The results also indicate that the Arg clusters may promote endocytosis and degradation of SMO, whereas multiple phosphorylation events neutralize the negative effect of the Arg clusters either by inhibiting endocytosis and/or promoting recycling of SMO (Zhao, 2007).

A striking feature of the SAID domain is that it contains multiple regulatory modules each of which consists of an Arg cluster linked to a phosphorylation cluster. The pairing of positive and negative regulatory elements may offer precise regulation, because phosphorylation at a given cluster may only neutralize adjacent negative element(s), leading to an incremental change in SMO activity. It is proposed that increasing phosphorylation gradually neutralizes the negative effect of multiple Arg clusters, leading to a progressive increase in SMO cell surface expression and activity. Thus, by employing multiple Arg clusters as inhibitory elements that are counteracted by differential phosphorylation, SMO acts as a rheostat to translate graded HH signals into distinct responses (Zhao, 2007).

G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling

The hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci). In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA). Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR), a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. This study presents in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. The results demonstrate that Smo functions as a canonical GPCR, which signals through Gαi to regulate Hh pathway activation (Ogden, 2008).

To examine whether a G protein is involved in Hh signalling, a series of G proteins was targetted by double-stranded RNA (dsRNA)-mediated knockdown. Drosophila clone-8 (Cl8) cells were treated with control or Gα-subunit-specific dsRNA and assayed for changes in Hh-mediated induction of a ptc-luciferase reporter construct. Whereas s (also called G-sα60A) and o (also called G-oα47A) dsRNAs do not significantly alter Hh-induced reporter activation knockdown is able to trigger a decrease in Hh-dependent reporter gene expression. Although not as effective as Smo knockdown in silencing Hh reporter gene activation, i (also called G-iα65A) dsRNA specific to the coding sequence, or 3' untranslated region (UTR), reduces Hh-induced reporter activity by approximately 70%, supporting a role for Gαi in the Hh pathway. To confirm the specificity of i dsRNA effects attempts were made to rescue reporter activity through ectopic expression of wild-type i or constitutively active GαiQ205L. Hh-stimulated reporter activity can be restored by both wild-type and constitutively active Gαi, confirming the specificity of the i dsRNA-mediated effects. Western blot analyses of Cl8 lysates reveal that cells treated with i dsRNA show attenuated stabilization of Ci and decreased Fu phosphorylation in response to Hh. Hh-induced Smo phosphorylation is maintained in the presence of i dsRNA, suggesting that Gαi functions downstream of Smo and upstream of Fu and Ci (Ogden, 2008).

To determine whether Gαi can modulate Hh pathway activity in vivo, i constructs were expressed in wing imaginal discs using MS1096-Gal4 or C765-Gal4. Expression of an inactive i mutant (iG204A) or wild-type i has little effect on wing vein patterning. However, expression of constitutively active iQ205L results in widening of longitudinal vein LV3-LV4 spacing and ectopic vein material on LV2 and LV3. The severity of this phenotype is dose-dependent, as higher-level expression of UAS-GαiQ205L triggers more severe ectopic vein material anterior to LV3, and further widening of LV3-LV4 spacing. Expression of iQ205L in wing imaginal discs also results in over-growth of the wing pouch, along with expansion of full-length Ci. This Ci expansion triggers ectopic expression of the Hh target gene decapentaplegic (dpp) in the wing pouch, as shown by a dpp-lacZ reporter gene. Gαi-mediated ectopic expression of dpp is consistent with the ectopic veins observed in wings expressing iQ205L. Taken together, these results support a role for activation of Gαi in regulating the stability of Ci, and link Gαi to regulation of a known Hh target gene (Ogden, 2008).

To determine whether Gαi functions downstream of Smo in vivo, the ability of Gαi to modulate Hh pathway activity was analysed in a smo sensitized background. As previously demonstrated, expression of a dominant-negative smo transgene, UAS-Smo5A, results in severe disruption of LV3-LV4 wing patterning. Expression of wild-type Gαi in this smo sensitized background allows for partial rescue of wing vein structures in the LV3/LV4 zone. Expression of constitutively active iQ205L results in a more complete rescue of the Hh loss-of-function phenotype, allowing for near total restoration of LV3/LV4 patterning. As a control, UAS-GFP was co-expressed with Smo5A, and found to have no effect on the Smo5A-induced phenotype (Ogden, 2008).

To examine the ability of GαiQ205L to modulate Ci stability and Hh target gene activation in the smo sensitized background, wing imaginal discs were immunostained with antibodies that recognize full-length Ci and the target gene product Ptc. UAS-Smo5A expression results in decreased ptc expression and disruption of the Ci gradient. Expression of constitutively active iQ205L in this smo sensitized background results in partial restoration of the Ci gradient and a near-complete rescue of ptc expression at the anterior/posterior border. These results support the model that Gαi contributes to the regulation of Hh target gene expression and Ci stability. Furthermore, the fact that this regulation occurs when Smo function is compromised suggests that Gαi affects Hh signalling at a level downstream of Smo (Ogden, 2008).

To determine whether Gαi is required for Hh signalling in vivo, Hh target gene expression was examined in clones of cells homozygous for i mutation. The null allele iP20 removes the entire coding region of the i gene, and is homozygous lethal. iP8 is a putative hypomorph, which removes the bulk of exons 1 and 2, but leaves the transcriptional start site intact and produces a transcript. Flies that are homozygous for the iP8 mutation are viable, but weak. Mosaic analysis reveals that expression of the Hh target gene dpp is decreased in both iP20 and iP8 mutant clones, supporting a role for Gαi in activation of Hh target genes in vivo. To confirm that the effects on dpp expression are due to loss of i, attempts were made to rescue iP20 null clones with UAS-i. Ectopic expression of i is able to rescue dpp reporter gene expression in iP20 clones, consistent with decreased dpp expression resulting from disruption of i (Ogden, 2008).

To determine whether compromised Gαi activity alters Hh-dependent patterning, the viable mutant allele iP8 and an additional viable allele described to be a null or strong hypomorph, i57 were used. Whereas homozygous iP8 and i57 mutants do not have vein fusions that are typical of strong Hh loss of function, their wings are smaller than wild-type wings. Small wing size might result from altered dpp expression in anterior cells of the wing pouch, as Dpp regulates wing blade size. Additionally, both iP8 and i57 mutant flies demonstrate varying degrees of incomplete thorax closure, as shown by mild to severe thoracic clefts. This phenotype is also consistent with decreased dpp expression, in that Dpp, in conjunction with JNK signalling, controls spreading of the anterior edge of wing imaginal discs to initiate thorax closure. To confirm that this phenotype results from decreased Hh signalling, ptc was expressed in the notum and dorsal compartment of the wing imaginal disc. ptc expression triggers the formation of a thoracic cleft when expressed under control of pannier and apterous promoters, suggesting that the thoracic phenotype observed in i flies results from compromised Hh signalling. Because iP20 null mutant animals are not viable, their wings or thoraces could not be examined. However, attenuation of Hh signalling by expressing dominant-negative Smo5A is enhanced in iP20 heterozygotes, as shown by disruption of LV3 (Ogden, 2008).

in vitro and in vivo data suggest that loss of Gαi might compromise Ci stabilization in Hh-receiving cells. When Ci and Smo levels were examined in i mutant clones, both appeared to be increased in a cell-autonomous manner. These results are consistent with the modest stabilization of Smo and Ci on in vitroi knockdown in non-Hh-treated cells. Although these results are unexpected, as Gαi loss is predicted to increase PKA activity and Ci degradation, previous studies have demonstrated that PKA functions to regulate Hh signalling both positively and negatively. Phosphorylation of Smo by PKA has a positive role in pathway activation, and might account for the modest stabilization of Ci that was observed (Ogden, 2008).

If Smo signals through Gαi it should be able to induce Gαi activation rapidly in response to Hh stimulation. To assay for Hh-mediated activation of Gαi, Cl8 cells were treated with conditioned media containing the amino-terminal Hh signalling molecule (HhN) or control conditioned media, then assayed for Hh-induced changes in intracellular cAMP. Within 5-10 min, HhN treatment reduces the basal intracellular cAMP concentration by approximately 50%. To confirm that the Hh-induced decrease in intracellular cAMP is dependent on Hh signalling through Smo and Gαi, cells were treated with smo, i or control dsRNA, then assayed for a Hh-induced decrease in cAMP. Whereas cells transfected with control dsRNA maintain the ability to decrease intracellular cAMP in response to HhN, cells transfected with either smo or i dsRNA are attenuated in their ability to do so. Taken together, these results support the idea that i is activated rapidly, in a Smo-dependent manner, in order to modulate cAMP levels in response to Hh (Ogden, 2008).

To determine whether modulation of cAMP can alter Hh signalling in vivo, a hypomorphic mutant allele of the cAMP-specific phosphodiesterase dunce (dnc1) was used to raise intracellular cAMP levels in a Hh-independent manner. Hemizygous dnc1 animals are viable with no obvious Hh defects. However, introduction of the dnc1 mutation into a smo sensitized background enhances the Smo loss-of-function phenotype, resulting in wings with near complete elimination of wing vein patterning. This enhanced Hh loss-of-function phenotype is similar to the phenotype obtained on decreasing smo gene dosage by one-half in the same smo sensitized background. Along with in vitro cAMP assays, these results indicate that Hh activates Smo to modulate intracellular cAMP, via Gαi, and that this function is important for proper pathway activity in vivo (Ogden, 2008).

Cos2 associates with membranes, microtubules, PKA, Smo, Fu and Ci. To determine whether Cos2 facilitates the coupling of Gαi with these Hh signalling components, lysates were prepared from cells expressing HA-Gαi, and then immunoprecipitated Cos2. It was found that Gαi associates with the Cos2 complex, and that this association is enriched in response to Hh. The binding of Fu to Cos2 is not altered by Hh, suggesting that the recruitment of Gαi to this protein complex is regulated. This result suggests that Cos2 facilitates the coupling of Smo with Gαi and additional downstream effectors necessary to transduce the Hh signal (Ogden, 2008).

This study has shown a requirement for Gαi in the Hh signalling pathway. Hh-mediated recruitment and activation of Gαi results in decreased intracellular cAMP, indicating that Hh may regulate PKA through modulation of the intracellular cAMP concentration. It was also demonstrated that Gαi can modulate Hh pathway activity in vitro and in vivo, and seems to do so at a level downstream of Smo. Furthermore, loss of Gαi alters Hh signalling in vivo, supporting the idea that Gαi is a requisite member of the Hh pathway (Ogden, 2008).

PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation

The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using an in vivo RNAi screen, PP4 and PP2A were identified as phosphatases that influence Hh signaling by regulating Smo and Ci, respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling activity. It was further found that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, it was shown that Hh downregulates Smo-PP4 interaction that is mediated by Cos2. Evidence is provided that PP2A is a Ci phosphatase. Inactivating PP2A regulatory subunit Widerborst (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, Wdb counteracts kinases to prevent Ci phosphorylation. Finally, evidence was obtained that Wdb attenuates Ci processing probably by dephosphorylating Ci. Taken together, these results suggest that PP4 and PP2A are two phosphatases that act at different positions of the Hh signaling cascade (Jia, 2009).

The screen used to identify the phosphatases differs from previous screens because an in vivo assay was used to examine Smo expression levels, which is a more direct readout, and because knockdown of specific phosphatase gene(s) involved in Smo dephosphorylation might not affect the pathway activity in a significant way and such gene(s) could have been missed in the previous RNAi screens with cultured cells. This study identified PP4 as a novel Hh signaling component that regulates Smo phosphorylation. The study provides the first evidence for the physiological Smo and Ci phosphatases, and uncovers the underlying mechanism of Smo regulation by phosphatase (Jia, 2009).

This study identified PP4 and PP2A to be negative and positive regulators in the Hh pathway, and it was shown that they exert their roles through Smo and Ci, respectively. Are PP4 and PP2A the only phosphatases in the Hh pathway? Although the data suggest that PP4 is a phosphatase for Smo, the possibility of the involvement of other phosphatase(s) cannot be excluded. Hh induces extensive Smo phosphorylation at numerous Ser/Thr sites, and multiple kinases are involved in these phosphorylation events. It might be possible that multiple phosphatases could be involved. In addition, loss-of-function studies on PP2A regulating Ci are not based on null mutations. This was due to the fact that genetic null mutations of the catalytic and regulatory subunits cause cell lethality. Thus, the results might not be exclusive (Jia, 2009).

Removal of PP4 by RNAi in wing discs induced Smo accumulation in A-compartment cells both near and away from the AP boundary. In addition, PP4 RNAi induced the elevation and anterior expansion of Hh target gene expression. However, the accumulated Smo caused by PP4 RNAi did not ectopically activate Hh target genes in cells away from the AP boundary. In addition, although Smo phosphorylation was potentiated by knocking down PP4 or abolishing Smo-PP4 interaction, the elevated phosphorylation did not suffice to promote Smo cell-surface accumulation. These data suggest that the basal phosphorylation of Smo regulated by PP4 is not sufficient to activate Smo, and that de novo Smo activation still depends on Hh (Jia, 2009).

Previous studies have shown that PKA and CK1 are required for Hh-induced Smo accumulation and signaling activity. Phosphorylation-deficient forms of Smo (with PKA or CK1 sites mutated to Ala) are defective in Hh signaling, whereas SmoSD123, the phosphorylation-mimicking Smo, has potent signaling activity and high level of cell-surface accumulation. Thus, the PKA and CK1 sites are apparently crucial in mediating Smo phosphorylation and activation. Hh treatment may cause increased phosphorylation at these sites. In addition to PKA and CK1 sites, there are many other Ser/Thr residues that are phosphorylated upon Hh stimulation. Although phosphorylation-mimicking mutations at these sites alone did not have discernible effect on Smo, their phosphorylation could modulate the cell-surface accumulation and activity of Smo phosphorylated at the three PKA/CK1 sites, which may at least in part explain why cell-surface accumulation and activity of SmoSD123 is still regulated by Hh. This study found that removing PP4 alone promoted Smo phosphorylation but did not elevate the cell-surface accumulation of Smo. It is possible that high levels of basal Smo phosphorylation in the absence of PP4 do not reach the threshold for promoting Smo cell-surface accumulation. It is also possible that basal Smo phosphorylation mainly occurs at sites other than the crucial PKA/CK1 phosphorylation clusters. In support of this notion, it was found that knockdown PP4 by RNAi promoted SmoSD123 to further accumulate on the cell surface in the absence of Hh (Jia, 2009).

How is Smo phosphorylation regulated? Hh may regulate Smo phosphorylation by regulating the accessibility of its kinase and/or phosphatase. In this study, it was found that Smo interacts with PP4 through amino acids 626-678, a region previously mapped to be a Cos2-interacting domain. It was further found that Smo-PP4 association diminished when Cos2 was knocked down by RNAi. A previous study revealed that Cos2 impedes Hh-induced Smo phosphorylation by interacting with amino acids 626-678 of Smo and Hh-induced phosphorylation of Cos2 at Ser572 dissociates Cos2 from amino acids 626-678 of Smo, thereby alleviating its inhibition on Smo phosphorylation. This study found that Cos2 inhibits Smo phosphorylation by recruiting PP4 and Hh promotes Smo phosphorylation by preventing Cos2-PP4 complex from binding to amino acids 626-678 of Smo. SmoDelta626-678, when not interacting with PP4, could still interact with Cos2 via a Cos2-interaction domain near the Smo C terminus. The Cos2-binding Smo C terminus might not recruit PP4. Taken together, these findings suggest that Hh may promote Smo phosphorylation at least in part by reducing the accessibility of a phosphatase (Jia, 2009).

Phosphorylation of Ci/Gli controls the balance of its activator and repressor activity. This study has demonstrate a role of PP2A in dephosphorylating Ci and attenuating Ci processing. However, it is not known whether Hh regulates PP2A to dephosphorylate Ci. Previous studies have shown that Hh interferes with the Cos2-Ci-kinase protein complex. It is possibly that Hh also regulates Ci phosphatase, or the accessibility of the phosphatase. Future studies should determine whether PP2A interacts with Cos2-Ci and whether such interaction is regulated by Hh (Jia, 2009).

Many aspects of Smo and Ci/Gli regulation are conserved across species. For example, both Drosophila and mammalian Smo proteins undergo a conformational switch in response to Hh stimulation. Ci/Gli proteolysis is mediated by the same set of kinases and E3 ubiquitin ligases. In addition, it has been shown that PP2A is involved in vertebrate Hh signaling, probably by regulating Gli nuclear localization and activity. Therefore, it would be interesting to determine whether PP4 and PP2A play similar roles in regulating phosphorylation of vertebrate Smo and Gli (Jia, 2009).

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila.

G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. This study provides evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding (Chen, 2010).

A genetic modifier screen for novel Hh signaling components identified Gprk2 as a positive regulator of Smo. Gprk2 was shown to be required for high but not low levels of Hh signaling activity. Evidence was provided that Gprk2 is a Smo kinase and that Gprk2 promotes maximal Smo activity by phosphorylating S741/T742 in Smo C-tail. Furthermore, a kinase-independent function of Gprk2 in Hh signaling was uncovered. Gprk2 forms a dimer/oligomer and binds Smo C-tail to promote the active state of Smo. Thus, this study reveals a novel mechanism for regulating a GPCR-like protein by GRK (Chen, 2010).

Previous studies suggest that Drosophila Gprk2 and mammalian GRK2 are involved in Smo phosphorylation because their knockdown in cultured cells either increased Smo mobility on SDS-PAGE or decreased metabolic labeling of Smo by γ-32p-ATP. However, these studies did not distinguish whether Gprk2/GRK2 phosphorylates Smo directly or indirectly through regulating other kinases. Neither did they reveal any biological relevance of Gprk2/GRK2-mediated phosphorylation in Hh signaling, since the relevant phosphorylation sites on Smo were not identified. In an in vitro kinase assay using purified substrates and a recombinant GRK, this study found that Smo is phosphorylated by GRK at S741/T742 and S1013/S1015. A mutagenesis study demonstrated that phosphorylation at S741/T742 is required for optimal Smo activation. Indeed, a previous study showed that Smo is phosphorylated at S741/T742 in cultured cells in the presence of Hh. In further agreement with the functional significance of S741/T742 phosphorylation, conserved S/T residues are found at the corresponding location in other insect Smo proteins (FlyBase) (Chen, 2010).

Interestingly, the in vitro kinase assay revealed that phosphorylation of S741/T742 by Gprk2 is regulated by PKA/CK1 phosphorylation at adjacent Ser residues, including S740, S743, and S746. Previous studies in mammalian systems suggest that GRKs tend to phosphorylate S/T residues embedded in an acidic environment. Phosphorylation at S740, S743, and S746 improves the acidic environment for S741/T742, which may account for the observed stimulation of S741/T742 phosphorylation by PKA/CK1. Indeed, mutating S740, S743, and S746 to Ala abolished PKA/CK1-mediated stimulation of S741/T742 phosphorylation, whereas converting these residues to acidic residues mimicked PKA/CK1-mediated stimulation. As Hh induces Smo phosphorylation by PKA and CK1, phosphorylation at S741/T742 by Gprk2 is likely to be stimulated by Hh in vivo (Chen, 2010).

Although phosphomimetic mutation at S741/T742 promotes Smo activity, it does not bypass the requirement for Gprk2 for optimal Smo activation because SmoSDGPSD failed to induce ectopic en expression in Gprk2 mutant discs. This implies that Gprk2 promotes Hh signaling through a mechanism in parallel to S741/T742 phosphorylation. It is possible that Gprk2 might act at an additional step downstream from Smo activation by phosphorylating intracellular Hh signaling components, or at the level of Smo activation by phosphorylating Smo at additional sites that have been missed by the in vitro kinase assay. However, the finding that the constitutively active form of Smo lacking the autoinhibitory domain (SAID: SmoΔ661-818) is insensitive to Gprk2 inactivation suggests that Gprk2 acts mainly at the level of Smo, although the possibility cannot be ruled out that Gprk2 may also play a minor role downstream from Smo. Interestingly, it was found that the kinase-dead form of Gprk2 (Gprk2KM) can rescue the activity defect of SmoSDGPSD in Gprk2 mutants, demonstrating that Gprk2 also regulates Smo in a phosphorylation-independent manner. The observation that Gprk2KM does not rescue the activity defect of SmoSD123 in Gprk2 mutants suggests that the phosphorylation-dependent and phosphorylation-independent mechanisms act in parallel rather than redundantly to promote Smo activation. Furthermore, evidence was obtained that Gprk2 interacts with the SAID independently of its kinase activity. Therefore, it is proposed that Gprk2 promotes Smo activation by counteracting Smo autoinhibition through binding to and phosphorylating the SAID (Chen, 2010).

At least two paralleled mechanisms have been attributed to Smo activation by Hh: (1) Smo cell surface accumulation, and (2) conformation change in Smo C-tail. Intriguingly, it was found that loss of Gprk2 resulted in increased rather than decreased Smo levels in cells that are not exposed to Hh or are exposed to low levels of Hh. However, unlike Hh stimulation, which preferentially stabilizes Smo on the cell surface, Gprk2 inactivation appears to stabilize Smo both inside the cell and on the cell surface. Furthermore, in the presence of high levels of Hh where Smo is accumulated at high levels on the cell surface, Gprk2 inactivation does not cause any discernible changes in either the level or subcellular distribution of Smo. Thus, the reduced Smo activity in Gprk2 mutant cells exposed to high levels of Hh is unlikely to be due to a change in Smo level or subcellular localization (Chen, 2010).

It is not clear what role Gprk2-mediated down-regulation of Smo levels might play in Hh signaling, although this may reflect an ancient mechanism by which GRK family kinases 'desensitize' GPCRs. In this regard, Gprk2-mediated down-regulation could serve as a mechanism to restrict the basal level of Hh signaling activity or to terminate or tune down Hh signaling activity once the Hh signal is withdrawn. However, this negative role of Gprk2 could be masked by its positive role. The mechanism by which Gprk2 down-regulates Smo levels remains unclear, although the kinase activity of Gprk2 appears to be required. Gprk2 could phosphorylate Smo and/or other proteins to promote Smo internalization and degradation. High levels of Hh could counteract Gprk2-mediated down-regulation of Smo by preventing Gprk2-meidated Smo internalization or by promoting Smo recycling (Chen, 2010).

FRET analysis provided strong evidence that Gprk2 is required for Smo to adopt and/or maintain its active conformation in response to Hh stimulation. A previous study suggested that Hh induces a conformational switch in Smo C-tail that is mediated by PKA and CK1 phosphorylation. In the absence of Hh, the Smo C-tail adopts a closed conformation in which the tail folds back, resulting in a close proximity between the C terminus and the third intracellular loop. The closed conformation is maintained, at least in part, through intramolecular electrostatic interactions between the multiple Arg clusters in the SAID and multiple acidic clusters near the C terminus. Hh-induced phosphorylation at PKA and CK1 sties disrupts the intramolecular electrostatic interactions, resulting in unfolding of the C-tail, which is reflected by a decreased intramolecular FRET (FRETL3C). In addition, phosphorylation promotes dimerization of two C-tails within a Smo homodimer, leading to increased proximity of the two C termini, as reflected by an increased C-terminal FRET (FRETC). Multiple intermediate conformational states may exist, depending on the levels of Smo phosphorylation, as increasing the number of phosphomimetic mutations progressively decreased FRETL3C and gradually increased FRETC. It was found that both an Hh-induced decrease in FRETL3C and an Hh-induced increase in FRETC were compromised by loss of Gprk2, suggesting that Gprk2 is critical for Smo to adopt and/or maintain the fully open conformation (Chen, 2010).

How does Gprk2 regulate Smo conformation? Genetic and FRET analyses demonstrated that Gprk2 promotes high levels of Hh signaling activity and regulates Smo conformation through both phosphorylation-dependent and phosphorylation-independent mechanisms. Furthermore, this study found that Gprk2 self-associates, binds the SAID, and promotes self-association of Smo C-tail. Interestingly, both Gprk2/SAID interaction and S741/T742 phosphorylation by Gprk2 are enhanced by PKA/CK1 phosphorylation. Taken together, the following model is proposed to account for the regulation of Smo conformation by Gprk2. In response to Hh, PKA/CK1-mediated phosphorylation of Smo C-tail promotes its unfolding and dimerization; however, in the absence of Gprk2, the open conformational state of Smo is unstable and may exist in equilibrium with the closed and/or partially open conformational states. Phosphorylation of Smo by PKA/CK1 promotes the binding of Gprk2 to the SAID and phosphorylation at S741/T742, both of which may stabilize Smo in the fully open and active conformation by preventing refolding of Smo C-tail and by 'cross-linking' the two C-tails within a Smo dimer via dimerization of Gprk2. In essence, Gprk2 may function as a 'molecular clamp' to promote the clustering of Smo C-tails. It is also possible that Gprk2 could cross-link two or more Smo dimers to form high-order oligomers, which might be essential for high levels of Hh signaling activity. This study thus reveals an unanticipated complexity in the regulation of Smo conformational states, and provides the first evidence that Smo conformation states are regulated by not only phosphorylation and intramolecular interactions, but also intermolecular interactions. It is possible that the closed conformation state of Smo is also regulated by intermolecular interactions in addition to intramolecular interactions. For example, it has been shown that Fu can directly bind the Smo C terminus in the absence of Hh, and this interaction may help stabilize the closed conformation of Smo C-tail. Indeed, disrupting Smo/Fu interaction led to increased basal activity of Smo (Chen, 2010).

Recent studies have emphasized the differences between vertebrate and Drosophila Hh signaling mechanisms. The sequence divergence between Drosophila and vertebrate Smo proteins and the lack of conserved PKA/CK1 phosphorylation sites in vertebrate Smo proteins have led to the proposal that vertebrate Smo proteins are activated through fundamentally distinct mechanisms. Nevertheless, a previous study revealed that Shh induces a conformational change in mSmo similar to that of dSmo, and forced clustering of mSmo also leads to pathway activation). GRK2 has been implicated as a positive regulator of the Shh pathway, and mSmo phosphorylation is affected by GRK2 silencing, although direct phosphorylation of mSmo by GRK2 has not been demonstrated. It is possible that GRK2 may substitute the role of PKA and CK1 and act as a major Smo kinase in vertebrates to promote the active Smo conformation. Alternatively, GRK2 may act in conjunction with other GRKs and/or yet-to-be-identified kinases to regulate Smo conformation, subcellular localization, and activity in vertebrates. The relatively weak phenotypes exhibited by GRK2 mutants are consistent with the latter possibility. This study also raised an interesting possibility that GRK2 may regulate mSmo not only by phosphorylation, but also by a kinase-independent mechanism such as a protein-protein interaction. Further investigation of the mechanism by which GRK2 and other kinases regulate mSmo will shed an important light on how vertebrate Smo activation is achieved (Chen, 2010).

Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses

In flies and mammals, extracellular Hedgehog (Hh) molecules alter cell fates and proliferation by regulating the levels and activities of Ci/Gli family transcription factors. How Hh-induced activation of transmembrane Smoothened (Smo) proteins reverses Ci/Gli inhibition by Suppressor of Fused (SuFu) and kinesin family protein (Cos2/Kif7) binding partners is a major unanswered question. This study shows that the Fused (Fu) protein kinase is activated by Smo and Cos2 via Fu- and CK1-dependent phosphorylation. Activated Fu can recapitulate a full Hh response, stabilizing full-length Ci via Cos2 phosphorylation and activating full-length Ci by antagonizing Su(fu) and by other mechanisms. It is proposed that Smo/Cos2 interactions stimulate Fu autoactivation by concentrating Fu at the membrane. Autoactivation primes Fu for additional CK1-dependent phosphorylation, which further enhances kinase activity. In this model, Smo acts like many transmembrane receptors associated with cytoplasmic kinases, such that pathway activation is mediated by kinase oligomerization and trans-phosphorylation (Zhou, 2011).

This study has shown that Fu is activated by phosphorylation in a Hh-initiated positive feedback loop and that Fu kinase activity alone can provoke the two key outcomes of Hh signaling in Drosophila, namely Ci-155 stabilization and Ci-155 activation. This previously unrecognized central thread of the Drosophila Hh pathway is strikingly similar to receptor tyrosine kinase (RTK) pathways or cytokine pathways, where the transmembrane receptor itself or an associated cytoplasmic tyrosine kinase initiates signal transduction via intermolecular phosphorylation. In Hh signaling, engagement of the Ptc receptor leads indirectly to changes in Smo conformation, and perhaps oligomerization that are relayed to Fu via a mutual binding partner, Cos2 (Zhou, 2011).

Three activation loop residues were identified as critical for normal Fu activity. Fu with acidic residues at T151 and T154 (Fu-EE) was not active at physiological levels in the absence of Hh but could initiate Fu activation in three different ways. First, increasing Fu- EE levels induces the full spectrum of Hh target genes and responses in wing discs and is accompanied by extensive phosphorylation, undoubtedly including S159, indicating that phosphorylation can fully activate Fu. Second, low levels of a Fu-EE derivative could synergize with an excess of wild-type Fu, provided the latter molecule had an intact activation loop and was kinase-competent, indicating that a feedback phosphorylation loop could initiate Fu activation even from a ground state containing no phosphorylated residues or their mimics. Third, Hh could activate Fu-EE or wild-type Fu, but this, unlike the above mechanisms, required Cos2 and the Cos2-binding region of Fu. Activation by Hh alters Smo conformation and increases the plasma membrane concentration of Smo-Cos2 complexes, suggesting that the role of activated Smo-Cos2 complexes may simply be to aggregate Fu molecules (Zhou, 2011).

In all of the above situations there is likely an important contribution of binding between the catalytic and regulatory regions of pairs of Fu molecules to allow cross-phosphorylation, as suggested by the impotence of the Fu-EE 1-305 kinase domain alone. The sites of inferred cross-phosphorylation, T151, S159, and S482 might most simply be direct Fu auto-phosphorylation sites but they may involve the participation of an intermediate kinase. Importantly, because Fu is the key activating stimulus and Fu is the key target for activation, there is no need to postulate additional upstream regulatory inputs into a hypothetical intermediary protein kinase. Phosphorylated residues in positions analogous to Fu S159 generally stabilize the active form of the protein kinase, whereas unphosphorylated residues at other positions, closer to the DFG motif may also, or exclusively, stabilize specific inactive conformations. By analogy, phosphorylated T151, T154, and S159 are likely to serve independent, additive functions, all of which are required to generate fully active Fu kinase. There are clearly additional phosphorylated residues on Fu, including the cluster at S482, S485, and T486. These residues are not essential for Hh or Fu-EE to generate fully active Fu when Fu is expressed at high levels. However, S485A/T486A substitutions did suppress activation of GAP-Fu in wing discs and in Kc cells, suggesting that stimulation of physiological levels of Fu, perhaps by lower levels of Hh uses S482, S485, and T486 phosphorylation to favor an active conformation of Fu or productive engagement of Fu molecules. Because the S482 region may be recognized directly as a substrate by the Fu catalytic site, this region may initially mask the catalytic site (in cis or in trans) and then reduce its affinity for the catalytic site once it is phosphorylated, permitting further phosphorylation of Fu in its activation loop (Zhou, 2011).

For a long time it was thought that Fu kinase acts only to prevent inhibition of Ci-155 by Su(fu), and Fu was postulated to accomplish this by phosphorylating Su(fu). This study mapped the sites responsible for the previously observed Hh- and Fu-stimulated phosphorylation of Su(fu) and showed that they were not important for regulating Hh pathway activity. It was found that CK1, like Fu, was required for Hh to oppose Su(fu) inhibition of Ci-155 and because each of the Fu-dependent phosphorylation sites in Fu and Su(fu) that were mapped in this study prime CK1 sites it is suspected that the critical unidentified Fu and CK1 sites for antagonizing Su(fu) will be found in the same molecule, with Ci-155 itself being a prime candidate (Zhou, 2011).

This study found that Fu does considerably more than just antagonize Su(fu). It was unexpectedly found that Fu kinase can also stabilize Ci-155 via phosphorylation of Cos2 on S572, which likely leads to reduced association of Cos2 Ci-155 activation independently of Su(fu), even when Ci-155 processing was blocked by other means (Zhou, 2011).

Some insight was gained into the key regulatory role that Fu plays in Hh signaling. The truncated partially activated Fu derivative, Fu-EE 1-473, exhibited constitutive activity when expressed at high levels but, unlike full-length Fu-EE, it was not activated by Hh. Importantly, a level of Fu-EE 1-473 expression could not be found in fumH63 mutant wing discs where Hh target genes were induced at the AP border but not ectopically. Hence, Hh regulation of Fu activity appears to be essential for normal Hh signaling. This contrasts with the normal Hh signaling observed in animals lacking Su(fu) and emphasizes that Fu is a key regulatory component that has essential actions beyond antagonizing Su(fu) (Zhou, 2011).

In mice, SUFU increases Gli protein levels and inhibits Gli activators in a manner that can be overcome by Hh, much as Su(fu) affects Ci levels and activity in flies. However, in mammalian Hh signaling there is no satisfactory mechanistic model connecting Smo activation and SUFU antagonism. This study found that mouse SUFU can substitute for all of the activities of Su(fu) in flies, including a dependence on both Fu and CK1 for Hh to antagonize silencing of Ci-155. These findings, and the observation that Drosophila Su(fu) can partially substitute for murine SUFU in mouse embryo fibroblasts, suggest that SUFU silencing of Gli proteins in mice is also likely to be sensitive to analogous changes in phosphorylation produced by at least one Hh-stimulated protein kinase. Even though the murine protein kinase most similar in sequence to Drosophila Fu is not required for Hh signaling at least three other protein kinases (MAP3K10, Cdc2l1, and ULK3) have been found to contribute positively to Hh responses in cultured mammalian cells. It will be of great interest to see if these or other protein kinases are activated by Hedgehog ligands, perhaps promoted by association with Smo-Kif7 complexes in a positive feedback loop, and whether they can antagonize mSUFU to activate Gli proteins, and perhaps even stabilize Gli proteins via Kif7 phosphorylation (Zhou, 2011).

Sequential phosphorylation of Smoothened transduces graded Hedgehog signaling

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. This study shows that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by PKA and further enhanced by casein kinase I (CKI). Protein phosphatase 1 (PP1) directly dephosphorylates PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylates PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. A functional link was established between sequentially phosphorylated Smo species and graded Hh activity. Thus, a sequential phosphorylation model is proposed in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling (Su, 2011).

The conversion of a gradient of the morphogen Hh into distinct transcriptional responses is essential for cell-fate decisions and tissue patterning during development. This study has provided genetic and biochemical evidence to support a model in which sequential phosphorylation of Smo, which is established by distinct kinases and phosphatases on specific serines, transduces graded Hh signaling. A basal extent of Smo activity, regulated by as yet unknown kinases and phosphatases, was sufficient to transduce low-threshold Hh signaling. PKA and PP1 collaborate to sustain PKA-phosphorylated Smo to transduce intermediate-threshold Hh signaling, whereas CKI and PP2A facilitate high-threshold Hh signaling by maintaining PKA-primed, CKI-phosphorylated Smo (Su, 2011).

Wdb-PP2A directly and specifically acts on CKI-pSmo to restrict high-threshold Hh signaling. Apart from PP2A, another phosphatase, PP1, specifically dephosphorylated PKA-phosphorylated Smo. This collaborative regulation between different phosphatases on the same substrate also functions in other cellular processes. For example, PP1 and PP2A dephosphorylate Par-3 to regulate cell polarity in the specification of neuroblast cell fate. Similarly, PP2A and PP4 respond to different DNA damage signals to dephosphorylate γ-H2AX to facilitate the repair of DNA double-strand breaks (Su, 2011 and references therein).

The activity of several Hh signaling components, including Smo, Ci, and Cos2, is regulated by phosphorylation. For example, PKA- and CKI-mediated phosphorylation of Ci leads to its destabilization. PP2A is implicated in regulating Ci activity in flies. This study confirmed that the catalytic PP2A subunit Mts associates with both Smo and Ci in cl-8 cells. Consistent with the substrate specificity of PP2A being conferred by its obligate regulatory subunits, this study found that Wdb specifically regulates the signaling potential of Smo. Another regulatory subunit, Tws, may direct PP2A activity toward Ci, which may potentially promote the translocation of CiFL to the nucleus, thereby activating Hh signaling. The use of distinct PP2A regulatory subunits in the same developmental process was also observed in transforming growth factor-β (TGF-β) signaling. The effect of the regulatory Bα subunit on PP2A activity activates Smad2 signaling, whereas the Bδ subunit inhibits Smad2 activity. The elaborate regulation of these two signaling systems by PP2A highlights a potential paradigm in which differential PP2A activity plays an essential role in developmental signaling. PP2A is a strong tumor suppressor; thus, modulation of PP2A activity provides an additional route by which development and tumorigenesis might be controlled (Su, 2011).

Another phosphatase, PP4, may play a role in inhibiting Smo; however, inhibiting PP4 alone is not sufficient to promote constitutive cell surface localization of wild-type Smo, unless Hh protein is provided. The surface localization of Smo is tightly linked to PKA- and CKI-dependent enhanced phosphorylation of Smo. PP4-specific RNAi further increases the extent of constitutive surface localization of Smo mutants that mimic PKA- and CKI-mediated phosphorylation, which suggests that PP4 may act on sites other than those in the PKA-CKI clusters (Su, 2011).

To delineate mechanisms whereby PP2A and PP4 might act on Smo, the expression of Hh signaling components as well as of Hh targets were systematically examined in wing discs expressing pp4 RNAi. In addition to the Smo stabilization, it was found that pp4 RNAi reduced the abundance of Cos2 protein. This might be as a consequence of the increased Smo abundance, because smo RNAi stabilized Cos2. Alternatively, PP4 might regulate Cos2 directly, because phosphorylated Cos2 is not stable. To distinguish between these two possibilities, the genetic relationship between smo and pp4 was examined by monitoring the stabilization of Cos2. Cos2 abundance was still reduced in wing discs containing both pp4 and smo RNAi. This effect is similar to the effect of pp4 RNAi alone, thus placing pp4 downstream of smo in regulating Cos2. Consistent with this, reduced expression of pp4 compromised Ptc and Collier/Knot (Col) expression at the AP boundary. The expanded area containing Ptc, albeit at a reduced abundance, away from the AP boundary has been observed previously. These experiments are consistent with a positive role of Cos2 in mediating maximal activation of Hh signaling in cl-8 cells as well as in wing discs; Ptc and Col expression are reduced in cos2 clones abutting the AP boundary. These data, together with the observation of a direct interaction between Cos2 and PP4 (Jia, 2009), argue that PP4 might also directly affect the extent of Cos2 phosphorylation (Su, 2011).

Smo contains three PKA-CKI phosphorylation clusters, with one PKA and two CKI consensus serines in each cluster. A previous study compared the signaling potential of phosphorylation-defective Smo by mutating PKA consensus serines to alanines in one, two, or three of the PKA-CKI clusters and concluded that at least six serines in Smo are required to fully induce the expression of ptc-lacZ, whereas only three serines are needed for the expression of dpp-lacZ. Another study further demonstrated that PKA- and CKI-mediated phosphorylation, which results in the generation of negatively charged residues, counteracts the positive charges conferred by nearby arginine clusters, thus enabling Smo to adopt a conformational change required to activate Hh signaling. These two studies support a model of collective Smo phosphorylation such that the identity of the phosphorylated serines in the PKA-CKI consensus clusters is probably not important; rather, the resulting negative charges collectively carried by these residues after phosphorylation are critical to determine the signaling strength of Smo (Su, 2011).

On the basis of this model, a variant Smo (Smo-CKI) in which the CKI, but not the PKA, consensus serines are mutated to alanines, thus rendering Smo-CKI resistant to CKI-mediated phosphorylation, would be anticipated to have the same signaling potential as Smo-PKA23, a variant containing a single intact PKA-CKI cluster, because both mutants contain three serines that can be phosphorylated. In smo loss-of-function clones, Smo-PKA23 is sufficient to drive expression of dpp-lacZ; however, Smo-CKI fails to rescue dpp-lacZ expression. The discrepancy between the effects of Smo-PKA23 and Smo-CKI on dpp-lacZ expression cannot be simply explained by the collective phosphorylation model. Moreover, these experiments reveal a functional distinction between different phosphorylated residues: Three PKA consensus serines in Smo-CKI may have less signaling activity than the single PKA and two CKI consensus serines in Smo-PKA23. The distinct signaling potentials of the two phosphorylation variants of Smo may be caused by different negative charge densities being carried by individual PKA-CKI clusters, or they may reflect intrinsic properties of sequential phosphorylation within each cluster (Su, 2011).

Indeed, the hierarchy of importance among individual PKA-CKI clusters in Smo has been revealed. Cluster 2 (also known as region V) is more prevalent than the other two clusters in activating ptc-luc reporter in smo-depleted cl-8 cells. Whether this functional distinction among PKA-CKI clusters also holds true in wing discs is unclear, because neither the hierarchical importance of individual clusters nor the relative importance of specific PKA and CKI phosphorylation events in neutralizing nearby arginine clusters has been directly studied. Nevertheless, when the relative importance of the three serines in cluster 2 was examined, the PKA-primed, CKI consensus sites (that is, sequential phosphorylation) were essential for Hh activation in cl-8 cells. The differential ability of SmoCKI-SA and SmoPKA-SA variants to activate dpp transcription in wing discs uncovered in this study is consistent with results obtained in cl-8 cells. Both observations challenge the model of collective Smo phosphorylation by arguing that the signaling potential of individual serines between each PKA-CKI cluster, as well as within a cluster, is most probably not equal. It is believed that regulated phosphorylation at specific serines may therefore contribute to graded Smo signaling (Su, 2011).

The collective phosphorylation model does not distinguish between the contributions of individual phosphorylated residues in PKA-CKI clusters. This study of phosphorylation-defective Smo variants revealed a Smo activity gradient in which phosphorylation at the PKA consensus sites and phosphorylation at the PKA-primed, CKI consensus sites were required for intermediate- and high-threshold Hh signaling, respectively. This activity gradient of Smo was directly visualized with the α-Smo-pS667 antibody. The abundance of PKA-phosphorylated Smo species, which is uncovered in Hh-stimulated fly cells by mass spectrometric analysis, increased initially but then declined sharply in response to Hh. As predicted from the model, phosphorylated Smo in response to intermediate-threshold Hh signaling was sensitive to dephosphorylation by PP1 but much less so to PP2A. Together, these data highlight the importance of sequential Smo phosphorylation to the transduction of graded Hh signaling. Sequential phosphorylation may be required to initialize graded Smo signaling activity. In addition, collective phosphorylation between different clusters may reinforce and maximize the Smo signaling potential to ensure the appropriate Hh signaling outcome (Su, 2011).

The presence of up to 26 serine or threonine residues in Smo that can be phosphorylated in response to Hh resembles the composition of residues found in the Kv2.1 potassium channel. Variable calcineurin-dependent dephosphorylation of Kv2.1 at 16 phosphorylated residues generates an activity gradient for channel gating and neuronal firing. The opposing actions of kinases and phosphatases on a multisite substrate are known through mathematical modeling to efficiently generate a range of stable phosphorylated forms. The spectrum of such distributions can be further increased with the number of phosphorylated sites. Two additional kinases, CK2 and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptor kinase 2 (GRK2), phosphorylate sites in Smo other than those targeted by PKA and CKI. Thus, the complex composition of phosphorylated residues in the cytoplasmic tail of Smo, coupled with versatile dephosphorylation by distinct phosphatases, provides an efficient and reliable mechanism to precisely convert the concentration thresholds of Hh into a graded signaling activity (Su, 2011).

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but understanding of the in vivo function and regulation of these enzymes is limited. This study investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described in this study identifies crucial roles for Tap42 phosphatase complexes in governing imaginal disc and fly development (Wang, 2012).

Understanding about the in vivo function of α4/Tap42, especially in development, is limited in part because global knockout of this gene in mice and flies leads to early embryonic death (see Cygnar, 2005 and Kong, 2004). Cellular studies have also revealed that depletion of α4/Tap42 causes death in embryonic stem cells, mouse embryonic fibroblasts, adipocytes, hepatocytes, B and T cells of the spleen and thymus, and Drosophila S2 cells (Bielinski, 2007; Kong, 2004; Yamashita, 2006). Although studies of a conditional (Cre-LoxP) α4 knockout in mouse hepatocytes and a mosaic assay of Tap42 in Drosophila wing disc have provided insights into the cellular biology of α4 and Tap42 (Cygnar, 2005; Kong, 2004), the impact of these gene products on the development of tissues and host have not yet been described. This report utilized Tap42-targeted RNAi and the Gal4/UAS system to investigate the biological effects of silencing Tap42 expression in specific Drosophila tissues. Suppressing the Tap42 gene using two tissue-specific drivers (pnr-Gal4 and ap-Gal4) led to a pleiotropic fly phenotype, which included major deformities in the adult thorax and wings as well as decreased survival rates. The experimental platform described in this study has allowed exploration of the role of Tap42 and Tap42-regulated phosphatases in the control of cellular signaling, tissue development, and Drosophila viability (Wang, 2012).

Analyses of Tap42RNAi wing discs revealed significant alterations in multiple signal transduction pathways including JNK, DPP, and HH. Marked increases in p-JNK signals were found in ap-Gal4>Tap42RNAi wing discs. This observation, together with previous studies showing increased c-Jun phosphorylation in α4-null mouse embryonic fibroblasts (Kong, 2004) and activated JNK in Tap42-depleted clones of fly wing discs (Cygnar, 2005), indicate that α4/Tap42 likely plays a negative role in regulation of JNK signaling. Silencing the Tap42 gene in the ap gene domain also changed DPP and HH signaling in the wing discs. Although ap-Gal4-mediated silencing of Tap42 had a profound effect on JNK, DPP, and HH signaling, these pathways were unaffected in pnr-Gal4>Tap42RNAi wing discs, thus demonstrating that the thorax cleft phenotype seen in the pnr-Gal4>Tap42RNAi flies is not due to alterations in these pathways. Collectively, these findings indicate that Tap42 plays a crucial role in the modulation of JNK, DPP, and HH signaling, but the effects of Tap42 on these pathways appear to play a minimal role in normal thorax development (Wang, 2012).

The HH pathway is one of the major guiding signals for imaginal disc development. Recent investigations have revealed that the phosphorylation state of Ci and Smo, two components of the HH signaling pathway, are controlled by Drosophila PP2A (Mts) and PP4 (Jia, 2009). Additional studies implicate a role for specific Mts complexes in the control of HH signaling, whereby holoenzyme forms of Mts containing the Wdb and Tws regulatory B subunits act at the level of Smo and Ci, respectively (Su, 2011). Together, these findings point to key roles for Mts and PP4 in HH signaling and suggest that a common subunit of these phosphatases, namely Tap42, may also be involved in HH signaling. Indeed, the current data clearly show that Tap42 plays an important regulatory role in this pathway as silencing of Tap42 within the wing discs leads to an elimination of both Smo and Ci expression. Although the precise role(s) of Tap42 in the control of HH signaling remains unclear, it likely involves Tap42-dependent regulation of one or more phosphatase catalytic subunits (e.g., Mts, PP4, and possibly PPV) or specific holoenzymes forms of these phosphatases (e.g., Wdb/Mts, Tws/Mts). The pleiotropic effects of Tap42RNAi on JNK, DPP, and HH signaling could be due to loss of Tap42's regulation of phosphatase activity, cellular levels, holoenzyme assembly, or subcellular localization (Wang, 2012).

Depletion of α4 in mouse embryonic fibroblasts caused an increase in phosphorylation of a variety of established PP2A substrates, which was attributed to a 'generalized defect in PP2A activity.' Instead of the expected unidirectional increase in protein phosphorylation, the current findings demonstrate a dual role for Tap42 in the control of JNK activation as hyperphosphorylation and hypophosphorylation of JNK were observed in the dorsal and ventral sides of the Tap42RNAi wing disc, respectively, relative to control wing discs. Silencing of Tap42 in the ap domain also impacted DPP in a bi-directional fashion; these flies exhibited significantly decreased DPP expression in the scutellum but augmented expression around the wing blade. Consistent with previous studies showing that PP2A functions at different levels within the Ras1 and HH pathways, the current data indicate that Tap42-regulated phosphatases likely target multiple substrates within the JNK and DPP pathways in different regions of wing discs (Wang, 2012).

Close examination of the PE cells in the wing disc revealed that Tap42 expression occurs in only a fraction of these cells. It is noteworthy that the majority of Tap42 localized in rows of cells delineating the PE/DP (peripodial epithelium/disc proper) boundary. These cells are commonly referred to as 'medial edge' cells, which represent a subpopulation of PE cells that play a crucial role in thorax closure during metamorphosis. Interestingly, α4-PP2A complexes appear to play a major role in the control of cell spreading, migration, and cytoskeletal architecture, presumably via their ability to modulate the activity of the small G-protein Rac. Yeast Tap42 has also been implicated in the cell cycle-dependent and polarized distribution of actin via a Rho GTPase-dependent mechanism. Therefore, it is hypothesized that the wing disc structural deformities and thorax cleft phenotype of Tap42RNAi flies are a result of unregulated phosphatases leading to defective spreading and migration of the medial edge cells during metamorphosis. The thorax cleft phenotype provides an opportunity to delineate the precise roles of Tap42-phosphatase complexes in processes controlling thoracic closure (e.g., cell spreading and migration) (Wang, 2012).

α4/Tap42 appears to function as an essential anti-apoptotic factor as cells lacking this common regulatory subunit of PP2A family members undergo rapid death. These studies implicate a role for α4/Tap42-dependent regulation of PP2A-like enzymes, and presumably the phosphorylation state of multiple pro- and anti-apoptotic proteins, in the maintenance of cell survival. The current findings reveal that silencing Tap42 in wing discs triggers apoptosis, thus providing supportive in vivo evidence that depletion of Tap42 (α4) leads to deregulated phosphatase action, which switches these enzymes from pro-survival to pro-apoptotic mediators. Because JNK activation is a hallmark feature of apoptosis, the overlap of apoptotic cells and hyperphosphorylated JNK indicates that the Tap42RNAi-induced apoptosis may be dependent on JNK activation (Wang, 2012).

Since α4 is required for maintaining the normal function of PP2A, PP4, and PP6, it is suspected that misregulation of these phosphatases could be responsible for the pleiotrophic phenotypes observed in Tap42RNAi flies. Consistent with this idea, introduction of the mtsXE2258 heterozygous allele into ap-Gal4>UAS-Tap42RNAi flies partially rescued the thorax and wing defects, and significantly improved fly survival rates. The partial rescue by mtsXE2258 suggests that the defects seen in the Tap42RNAi flies are due, in part, to unregulated Mts activity, possibly as a result of increased Mts levels or enzymatic activity. Indeed, previous studies have demonstrated an accumulation of Mts in Tap42-depleted clones of the fly wing disc. Thus, mtsXE2258 appears to function as a mild mutant that partially restores misregulated Mts function following depletion of Tap42. However, given the biochemical findings showing that Tap42 also interacts with PP4 and PPV, additional studies will be needed to determine the relative contribution of these phosphatases to the Tap42RNAi-induced defects (Wang, 2012).

The phenotypes observed in flies expressing Tap42RNAi could also be attributed to loss of a phosphatase-independent function(s) of Tap42 that controls normal fly development. However, introduction of a phosphatase binding-defective mutant of Tap42 (Tap42ED) into the Tap42RNAi background failed to rescue the phenotypes and lethality associated with Tap42 depletion. In contrast to Tap42ED, introduction of Tap42WT fully rescued the phenotypes and lethality of the Tap42RNAi flies. These findings indicate that the Tap42RNAi-induced phenotypes are entirely due to the impaired interactions between Tap42 and PP2A family members, and provide compelling support for the hypothesis that Tap42-dependent regulation of the functions of these enzymes is crucial for normal wing disc development and Drosophila viability (Wang, 2012).

Although understanding the exact molecular mechanisms underlying Tap42's regulation of PP2A family members is still incomplete, these studies clearly demonstrate that Tap42-phosphatase interactions play crucial roles in the control of multiple signaling pathways governing cell growth and survival. The experimental platform described in this report will undoubtedly serve as a valuable system to further explore the in vivo function and regulation of Tap42-phosphatase complexes. Furthermore, given the remarkable phenotypes seen in the Tap42RNAi flies (e.g., thorax cleft and deformed wings), it is anticipated that this model system will drive future studies (e.g., phenotype-based suppressor/enhancer screens) aimed at identifying direct targets of Tap42-regulated phosphatases, as well as additional pathways under the control of these phosphatase complexes (Wang, 2012).

Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. This study demonstrates that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, this study found that the non-visual beta-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, it was shown that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, these results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation (Li, 2012).

Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling

The Hedgehog (Hh) signaling pathway plays a important role in metazoan development by controlling pattern formation. Malfunction of the Hh signaling pathway leads to numerous serious human diseases, including congenital disorders and cancers. The seven-transmembrane domain protein Smoothened (Smo) is a key transducer of the Hh signaling pathway, and mediates the graded Hh signal across the cell plasma membrane, thereby inducing the proper expression of downstream genes. Smo accumulation on the cell plasma membrane is regulated by its C-tail phosphorylation and the graded Hh signal. The inhibitory mechanism for Smo membrane accumulation in the absence of Hh, however, is still largely unknown. This study reports that Vps36 of the ESCRT-II complex regulates Smo trafficking between the cytosol and plasma membrane by specifically recognizing the ubiquitin signal on Smo in the absence of Hh. Furthermore, in the absence of Hh, Smo is ubiquitylated on its cytoplasmic part, including its internal loops and C-tail. Taken together, these data suggest that the ESCRT-II complex, especially Vps36, has a special role in controlling Hh signaling by targeting the membrane protein Smo for its trafficking in the absence of Hh, thereby regulating Hh signaling activity (Yang, 2013).

As one of essential membrane proteins of the Hh signaling pathway, Smo contributes to transducing the graded Hh signal across the cell plasma membrane. Accumulation of Smo on the cell plasma membrane and its C-tail phosphorylation are finely controlled by the Hh signal. In the absence of Hh, Smo fails to accumulate on the cell plasma membrane. In the presence of Hh, Ptc relieves its inhibition of Smo activity by binding Hh, resulting in Smo cell plasma membrane accumulation. However, it remains largely unknown how Smo trafficking is regulated during Hh signaling. This study found that the ESCRT-II complex regulates Smo trafficking in the absence of Hh, through the interaction between Vps36 and the ubiquitylated Smo. in vitro and in vivo observations show that Vps36 specifically recognizes Smo and then regulates its endosome trafficking. It was also found that the recognition of Smo by Vps36 is dependent on multiple ubiquitylation sites of its C-tail and internal loops. Meanwhile, Smo association with Vps36 is regulated by its C-tail phosphorylation and Hh signal. Moreover, the GLUE domain of Drosophila Vps36 is involved in recognizing ubiquitin, as does its mammalian homolog. However, in this experiment, it was also observed that loss of Hrs, a component of the ESCRT-0 complex, results in Smo intracellular endosome accumulation. This also implied that, unlike Hrs, Vps36 might play a specific role in Smo regulation (Yang, 2013).

Ubiquitin can function as a signal for the endosome sorting and trafficking of many membrane proteins. This study reports that the Hh signal regulates Smo trafficking by controlling its ubiquitylation. In the absence of Hh, Smo was ubiquitylated and recognized by ESCRT complexes for endosome trafficking and for further sorting, therefore inhibiting its accumulation on the cell plasma membrane and blocking its activity. However, in the presence of Hh, the ubiquitylation of Smo is inhibited, and the cytosol to plasma membrane trafficking of Smo is blocked, resulting in its accumulation on the cell membrane and activation of the Hh pathway (Yang, 2013).

The SAID domain of Smo is important to its recognition by Vps36, which is required for Smo trafficking. The Smo SAID domain has three PKA and CKI phosphorylation clusters and four arginine motifs, which are very important for Smo activity regulation and its conformational change in response to Hh. A recent study suggested that Smo is ubiquitylated at multiple lysine residues in the SAID domain (Li, 2012). To characterize Smo ubiquitylation, all 13 lysine residues located in the SAID domain (SmoK13R) were mutated. Unexpectedly, it was found that SmoK13R, in which all lysine residues in the SAID domain were mutated to arginine, showed equivalent ubiquitylation levels compared with SmoWT. To further address this question, more lysine residues in Smo cytoplasmic regions were mutated, including its C-tail and internal loops. Finally, it was found that only overexpression of SmoKallR, in which all the lysine residues in the cytoplasmic part were mutated to arginine, leads to a dramatic decrease of the Smo ubiquitylation level, and thereby its recognition by Vps36. One explanation for this phenomenon is that Smo ubiquitylation sites are not limited to only a few lysine residues. It is likely that the Hh signal regulates Smo trafficking by controlling Smo ubiquitylation on multiple lysine sites. Meanwhile, even SmoKallR still has basal level ubiquitylation. At this stage, the possibility cannot be ruled out that, in the absence of Hh, other amino acid residues, such as cysteine and serine residues, can be ubiquitylated (Yang, 2013).

Collectively, this work uncovered a mechanism whereby the Hh signal controls Smo trafficking by regulating Smo ubiquitylation. The next question would be how the Hh signal regulates Smo ubiquitylation. One hypothesis is that the Hh signal induces Smo phosphorylation and conformational change, which results in an 'open' and activated conformation of Smo that fails to recruit E3 ubiquitin ligase for ubiquitylation. Consistent with this notion, the SAID domain of Smo, which is important for the maintenance of Smo 'closed' conformation by arginine motifs, has been found to be critical for Smo cell plasma membrane localization. This study further found that Smo lacking the SAID domain had a lower ubiquitylation level independent of the ubiquitylation of lysine residues in this domain. It is also possible that the binding of E3 ubiquitin ligase to Smo is regulated by the Hh signal and Smo C-tail phosphorylation. In addition, the results suggest that Smo trafficking is regulated by the ESCRT-II complex only in the absence of Hh. How Smo internalization is regulated in the presence of Hh, as well as the specific E3 ligase and components involved in this process warrants further study (Yang, 2013).


smoothened: Biological Overview |Evolutionary homologs | Developmental Biology | Effects of Mutation | References

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