smoothened
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 tra