cAMP-dependent protein kinase 1


Disruptions of a Drosophila gene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIbeta (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles show specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, are normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads reveal a complex lesion of the PKA-RI locus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis reveals reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP is significantly higher than normal in homogenates from mutant adult heads. Two mutant alleles fail to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning (Goodwin, 1997).

A unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene has been identified in Drosophila with a severely hypomorphic if not null mutation: pka- RIIEP(2)2162. Extracts from pka- RIIEP(2)2162 flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka- RIIEP(2)2162 shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka- RIIEP(2)2162 is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol (Park, 2000).

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened (Smo) 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).

Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus

A 75-kDa proteolytic product of the full-length Cubitus interruptus (Ci) protein translocates to the nucleus and represses the transcription of Ci target genes. In cells that receive the hh signal, the proteolysis of Ci is inhibited and the full-length protein can activate the hh target genes. Because protein kinase A (PKA) inhibits the expression of the hh target genes in developing embryos and discs and the loss of PKA activity results in elevated levels of full-length Ci protein, PKA might be involved directly in the regulation of Ci proteolysis. It has been demonstrated that the PKA pathway antagonizes the hh pathway by phosphorylating Ci (Chen, 1998).

Mutant Ci proteins that cannot be phosphorylated by PKA have increased transcriptional activity compared with wild-type Ci. A search for the RRXS/T consensus PKA phosphorylation motif in Ci shows that there are four consensus PKA phosphorylation sites clustered in the C-terminal transactivation domain. To determine whether PKA affects CI transcriptional activity directly, the serines in the PKA sites were mutated to alanines either singly [m1 (Ser to Ala at aa 838), m2 (Ser to Ala at aa 856), m3 (Ser to Ala at aa 892), m4 (Thr to Ala at aa 1,006)] or in various combinations, including the mutation of all four sites (null). These PKA target site mutants were cotransfected with a Ci target reporter construct and the CAT activities were compared with those of wild-type CI. M1, m2, m3, and the mutation that is null for all four PKA sites greatly stimulates CI activity, whereas m4 has less of an effect. This result suggests that the inhibition of phosphorylation of any one of the first three CI PKA sites is sufficient to stimulate CI transcriptional activity. Although the major effect of PKA activity in this system is negative, when Ci can no longer be the target for PKA phosphorylation, a minor positive effect of PKA has been unmasked. PKA might phosphorylate other factors crucial for CI-mediated transactivation, such as dCBP, to activate CI-mediated transcription. This dual role played by PKA is in agreement with the genetic result by Ohlmeyer (1997) that overexpression of the PKA catalytic subunit increases the hh target gene expression in a ci- and smo-dependent manner (Chen, 1998).

The PKA-mediated phosphorylation of Ci promotes its proteolysis from the full-length active form to the 75-kDa repressor form. Kc cells were transfected with HA-CI (WT) or HA-CI (null) (Ci cDNA fused to an N-terminal HA tag) and the exogenous HA-CI protein was precipitated from whole-cell extracts by a rat monoclonal anti-HA antibody. The HA antibody detects both the 155-kDa full-length form and the 75-kDa form when HA-CI wild type is transfected into Kc cells. When HA-CI (null), which has all four PKA sites mutated, was transfected into cells, the 75-kDa CI protein could not be detected, indicating that PKA phosphorylation of CI is required for the proteolysis of CI. Kc cells were also cotransfected with HA-CI (WT) or HA-CI (null) and either PKA or PKI, an inhibitor of PKA. Cotransfection of PKI with HA-CI (WT) inhibits the formation of the 75-kDa CI protein, whereas the cotransfection of HA-CI (WT) and PKA stimulates the formation of the 75-kDa CI product This result suggests that the phosphorylation of CI by PKA stimulates the proteolytic processing of CI in Kc cells (Chen, 1998).

Drosophila CBP has several consensus PKA sites, one of which is located between the third and fourth zinc fingers and is well conserved among members of the p300/CBP gene family. Results from other research has shown that a GAL-CBP1678-2441 fusion protein, which includes this conserved PKA site, is a transcription activator and that the activity of this chimera increases when PKA activity is stimulated in PC12 cells; however, there is no evidence as yet that PKA increases CBP activity by phosphorylation of this site. In addition, the finding that the activity of GAL-CBP1-460 is greatly stimulated by PKA treatment in PC12 cells (17-fold) further supports the idea that PKA modulates the activity of CBP. However, the current results do not rule out the possibility that PKA phosphorylates and activates other transcriptional factors involved in CI-mediated gene activation as well. Whether the hh-smo pathway directly affects PKA function is unclear (Chen, 1998).

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that Ci exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. Exogenous expression of Ci that is mutant for four consensus PKA sites, CI(m1-4), causes ectopic expression of wingless in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not Ci(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of Ci(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of Ci. PKA is shown to directly phosphorylate the four consensus phosphorylation sites in vitro. Taken together, these results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of Ci phosphorylation (Chen, 1999).

It can be determined whether PKA phosphorylates consensus PKA target sites in vitro. Ci fragments of wild type Ci and of CI(m1-4) that contain the four PKA sites (aa441-1065) were fused to GST. Two-dimensional tryptic phosphopeptide maps of the expressed fusion proteins were generated. There are at least 13 phosphopeptides that are labeled by PKA in the wild-type Ci peptide. In vitro, PKA can recognize RxS/T, the subset RRxS/T, RxxS/T and RKxxS/T. The phosphorylation of S is preferred 40:6 over T and in vivo, the RRxS site is preferred 2:1 over the others. The four consensus RRxS/T sites in Ci were chosen for mutation because they would probably be the preferred phosphorylation sites in vivo. Scanning the Ci fragment for all possible consensus PKA sites, it was found that all of the phosphopeptides can be accounted for by the number of PKA consensus sites in the fusion protein. Three of the strong spots and two weaker spots that are present in the wild-type fragment are missing in the mutant fragment, demonstrating that PKA can specifically and directly phosphorylate the four RRxS/T consensus PKA sites in vitro. The two weak spots are difficult to distinguish and may represent only one spot or incomplete digestion of a single peptide. GST alone was not phosphorylated (Chen, 1999).

What of the positive regulation of Ci activity by hh? Because the genetic data suggests that hh does not regulate PKA directly, it may be that hh affects the phosphorylation state of Ci by activating a phosphatase, or through changing the accessibility of Ci to a phosphatase. In support of this idea is the observation that the phosphatase inhibitor, okadaic acid, stimulates Ci proteolysis, even in the presence of a HH signal. HH signaling stimulates fu kinase activity to transform full-length Ci to a transcriptional activator. It may also be that fu activity renders full-length Ci inaccessible to PKA phosphorylation (Chen, 1999).

Proteolysis of Cubitus interruptus in Drosophila requires phosphorylation by Protein Kinase A

The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. A mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically alters levels of full-length wild-type Ci in embryos and imaginal discs, but does not significantly alter full-length Ci5m levels. These results are corroborated by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. It is therefore proposed that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. It is also showm that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step (Price, 1999).

How does PKA phosphorylation of Ci-155 lead to its proteolysis? Loss of cos2 activity in wing disc clones induces high levels of Ci-155, suggesting that the integrity of the multiprotein cytoplasmic complex that contains Ci or the association of this complex with microtubules may be necessary in order for proteolysis to occur. It is possible that Ci phosphorylation also affects proteolysis by altering these interactions. A more direct role for PKA phosphorylation of Ci has been proposed based on the sequence and properties of the (Supernumerary limbs) Slimb protein, which affects the conversion of Ci-155 to Ci-75. Slimb belongs to a family of F-box/ WD40-repeat proteins implicated in binding to and targeting phosphorylated molecules for ubiquitin-mediated degradation. It was recently shown that the vertebrate Slimb homolog, beta-TRCP, targets IkappaB and beta-catenin for ubiquitin-mediated degradation by binding specifically to a phosphorylated motif (DSGXXS, where both serines must be phosphorylated) present in both proteins. Whether Slimb participates in such a direct manner in Ci proteolysis is not clear. Slimb has not been shown to bind to Ci, and Ci proteolysis has not been shown to involve ubiquitination; Ci proteolysis is also unusual in being incomplete, leaving a stable 75 kDa product. Sequences around three Ci sites show some extended similarity to each other but are quite different from the IkappaB and beta-catenin consensus. It will be interesting to determine if Slimb, or another F-box protein, can bind directly to these regions of Ci when phosphorylated by PKA. Since Slimb recognition requires phosphorylation at multiple residues and the PKA site consensus in Ci contains additional serines, it is worth considering that the activity of another protein kinase in addition to PKA may also contribute to the regulation of Ci proteolysis (Price, 1999 and references).

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

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

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

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

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

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

Genetic evidence for a protein kinase A/Cubitus interruptus complex that facilitates processing of Cubitus interruptus in Drosophila

Hedgehog (Hh) activates a signal transduction pathway regulating Cubitus interruptus (Ci). In the absence of Hh, full-length Ci (Ci-155) is bound in a complex that includes Costal2 (Cos2) and Fused (Fu). Ci-155 is phosphorylated by protein kinase A (PKA), inducing proteolysis to Ci-75, a transcriptional repressor. Hh signaling blocks proteolysis and produces an activated Ci-155 transcriptional activator. The relationship between PKA and the Ci/Cos2/Fu complex is unclear. PKAc is generally thought to be part of a PKA holoenzyme where its activity is inhibited by PKAr and regulated by cAMP. Since Hh signaling leads to inhibition of Ci-155 phosphorylation and proteolysis, one might expect that this would coincide with a drop in ambient cAMP level and inhibition of PKAc activity by PKAr. No change in cAMP levels, however, has been detected in a mouse cell line sensitive to Sonic hedgehog signaling when exposed to Sonic hedgehog. Experiments addressing this question in Drosophila have employed ectopic expression of PKAc*, a constitutively active mutant mouse catalytic subunit impaired in interaction with PKAr. Low levels of PKAc* expression appear to not affect normal Hh signaling, while high levels override Hh signaling and promote Ci-155 proteolysis (Kiger, 2001a).

Ectopic expression of the PKAc inhibitors PKAr* and PKI(1-31) produces quite different responses from Hh target genes: PKAr* mimics the effect of mitotic clones deficient in PKAc (homozygous for loss-of-function DC0 mutants), while PKI(1-31) has no effect, despite its demonstrated ability to inhibit endogenous PKAc. The basis for the different effects of PKAr* and PKI(1-31) has been addressed through studies of mutant forms of PKAr* and PKAc. The possibility that free PKAr* (and free wild-type PKAr) has a target other than PKAc through which it induces Hh signaling is tested. Precedence for such a role can be found in Dictyostelium, where free PKAr binds to, and activates, a cAMP-specific phosphodiesterase. In this view, a mitotic clone deficient in PKAc would induce Hh signaling because of the free PKAr created therein. It has been shown that ectopic expression of a catalytic site mutant of PKAc, PKAcA75, produces an unexpected bipartite phenotype compared to that of ectopic PKAc. To wit, PKAcA75 is null with respect to PKAc phenotypes and is dominant negative with respect to activation of Hh target genes. These observations cast new light on the role of PKAc in Hh signaling and indicate that PKAc is part of a larger complex that includes Ci-155. This insight makes it possible to understand those attributes of PKAr*, lacking in PKI(1-31), that allow PKAr* to block PKAc involved in phosphorylation of Ci-155 (Kiger, 2001a).

Thus, an examination was performed of Hh target gene expression caused by mutant forms of PKA regulatory (PKAr) and catalytic (PKAc) subunits and by the PKAc inhibitor PKI(1-31). The mutant PKAr*, defective in binding cAMP, has been shown to activate Hh target genes solely through its ability to bind and inhibit endogenous PKAc. Surprisingly, PKAcA75, a catalytically impaired mutant, also activates Hh target genes. To account for this observation, it is proposed that PKAc phosphorylation targeting Ci-155 for proteolysis is regulated within a complex that includes PKAc and Ci-155 and excludes PKI(1-31). This complex may permit processive phosphorylation of Ci-155 molecules, facilitating their processing to Ci-75. Phosphorylation of a number of sites on Ci-155 is required for its proteolysis. If so, Ci-155 must be able to rotate to access the active site of the bound PKAc and other components of the complex must be required to hold it in place. Such steric constraints could prevent PKI(1-31) from finding its PKAc binding site, explaining the inability of this inhibitor to induce Hh target gene expression (Kiger, 2001a).

Determining what other components make up the PKAc/Ci complex will be an important step in understanding Hh signal transduction. Since most cytoplasmic Ci-155 is bound in a Ci/Cos2/Fu complex, PKAc must be part of this complex or part of another complex that is closely tied to it. The association of PKAr type I and PKAc in vitro is exquisitely balanced by the binding of MgATP to the holoenzyme at the PKAc catalytic site. A decrease in MgATP concentration leads to rapid dissociation of the holoenzyme. Since PKAcA75 is mutant at a residue required for binding of MgATP, it seems probable that PKAcA75 is unable to form a holoenzyme with PKAr type I, just as it is unable to block phosphorylation of most PKAc substrates. Indeed, the fact that PKAr*, a type I subunit, is less effective than PKAcA75 in inducing Hh target genes could be due to PKAr type I not being part of the PKA/Ci complex. On the other hand, binding of PKAr type II to PKAc does not require MgATP, suggesting that PKAcA75 may enter the complex bound to PKAr type II. If PKAr type II is part of the complex, then the presence of an A-kinase anchor protein (AKAP) might also be expected. At least five AKAPs have been described in Drosophila that tether PKA type II to plasma membranes or the cytoskeleton. It is also possible that an inhibitor protein might escort PKAc to the complex but not be part of the complex (Kiger, 2001a).

Knowing the components of the complex will permit choices to be made between possible models. In one model, in the absence of a Hh signal, ambient cAMP level in the complex would be high enough to dissociate PKAr and PKAc and permit processive phosphorylation of Ci-155. It is envisioned that dissociated PKAr and PKAc would remain bound within the complex. A Hh signal then might act to reduce cAMP level and allow bound PKAc and bound PKAr to reassociate, preserving Ci-155 from phosphorylation. In another model, active PKAc may be bound within the complex, and a Hh signal might activate a phosphatase to convert phosphorylated Ci-155 into a transcriptional activator. Whether or not cAMP is involved in Hh signaling remains an open question (Kiger, 2001a).

Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen synthase kinase 3 and Casein kinase 1

The secreted signaling molecule Hedgehog regulates gene expression in target cells in part by preventing proteolysis of the full-length Cubitus interruptus (Ci-155) transcriptional activator to the Ci-75 repressor form. Ci-155 proteolysis depends on phosphorylation at three sites by Protein Kinase A (PKA). These phosphoserines prime further phosphorylation at adjacent Glycogen synthase kinase 3 (GSK3) and Casein kinase I (CK1) sites. Alteration of the GSK3 or CK1 sites prevents Ci-155 proteolysis and activates Ci in the absence of Hedgehog. Ci-155 proteolysis is also inhibited if cells lack activity of the Drosophila GSK3. Conversely, Ci-155 levels are reduced in Hedgehog-responding cells by overexpression of PKA and the Drosophila CK1, Double-time, a regulator of circadian rhythms. Thus Shaggy/GSK3 is implicated in the functioning of the Hedgehog pathway, in addition to its well known role in the Wingless pathway (Price, 2002).

Phosphorylation of Ci at three defined PKA sites primes further phosphorylation at adjacent GSK3 and CK1 sites. This PKA-primed phosphorylation could be catalyzed by purified mammalian GSK3ß and CK1delta enzymes or by activities in Drosophila embryo extracts. Changing the target serines of either GSK3 or CK1 consensus sites to alanines prevents proteolysis of Ci-155 to Ci-75 in flies. This result was demonstrated both by Western blots of embryo extracts and by assaying for the activity of Ci-75 as a transcriptional repressor in wing imaginal discs. It is argued that the resistance of these altered Ci molecules to proteolysis results from altered phosphorylation rather than a change in amino acid identity per se, because elimination of Sgg GSK3 activity produces a similar result and because the PKA sites required for priming further phosphorylation must themselves be intact in order for Ci-155 to be proteolyzed to Ci-75 (Price, 2002).

How extensively must Ci be phosphorylated in order to be proteolyzed? Whether each potential PKA, GSK3, or CK1 phosphoacceptor site is essential for Ci proteolysis has not been tested in flies. Evidence obtained in tissue culture cell studies suggests that Ci proteolysis is largely inhibited by alteration of single PKA sites, and at least one PKA site (site 1) is critical in flies. Alteration of two GSK3 sites, adjacent to PKA sites 2 and 3, prevents Ci-155 proteolysis. Hence, the view is favored that each of the phosphorylation sites in this region of Ci contributes significantly to Ci-155 proteolysis (Price, 2002).

Inhibition of the 26S proteosome in clone 8 tissue culture cells leads to the accumulation of highly phosphorylated full-length Ci forms of lowered gel mobility, especially if phosphatase activity is also inhibited. Ci-155 from untreated clone 8 cells can be separated into about six isoforms by isoelectric focusing. The location of phosphorylated residues was not mapped in either of these studies. However, it appears that Ci phosphorylated on a small number of sites, perhaps largely PKA sites, is stable enough to visualize, whereas subsequent, perhaps cooperative, phosphorylation, most likely on adjacent GSK3 and CK1 sites, leads to rapid Ci-155 proteolysis and is therefore evident only if proteolysis is artificially inhibited (Price, 2002).

The basic arrangement of PKA sites flanked by PKA-primed GSK3 and CK1 sites is conserved in Gli2 and Gli3 for each of the three PKA sites in Ci, with an additional fourth motif between PKA sites 2 and 3 of Ci. The identity of amino acids in each cluster extends beyond the consensus SGSK3RRXSPKAXXSCK1. For instance, PKA site 1 has an adjacent CK1 site followed by a second CK1-primed site (RRXSPKAXXSCK1XXSCK1), but there are no GSK3 sites. PKA sites 2 and 3 are flanked by GSK3 sites and CK1 sites (SGSK3RRXSPKAXXSCK1), but only site 3 includes a second GSK3 site (SGSK3XXXSGSK3RRXSPKA). Ignoring the possibility of additional interstitial phosphorylations in this region due to GSK3 priming of CK1 sites and vice versa, Ci contains a total of eight PKA-primed GSK3 or CK1 phosphorylation sites, whereas Gli2 and Gli3 contain eleven and nine, respectively, in this region of less than 80 amino acids. Gli1 has only two PKA sites in this region with three associated CK1 sites and only one GSK3 site. Commensurate with sequence conservation, both Gli2 and Gli3 appear to be proteolyzed when expressed in Drosophila, whereas Gli1 remains full-length. Processing of Gli proteins in flies appears to correspond, at least approximately, to their fate in their normal environment. These data are consistent with the proposal that a conserved mechanism of PKA-dependent proteolysis of Ci/Gli proteins depends on creating highly phosphorylated clusters of regularly spaced phosphoserine residues (Price, 2002).

How do multiple phosphorylations of Ci target it for degradation? Paired GSK3 phosphorylation sites are crucial for recognition of ß-catenin by Slimb/ß-TrCP, but they fall within a more specific consensus sequence DS(P)GXXS(P) that is conserved in IkappaB. None of the GSK3 or, of course, the CK1 or PKA sites in Ci conform to this consensus. It is possible that Slimb/ß-TrCP recognizes more epitopes than currently appreciated or that the presence of multiple weak binding sites collectively contributes to association with Slimb. The latter mechanism has been demonstrated for the recognition of yeast Sic1, which is phosphorylated within multiple suboptimal binding sequences, by the F box protein Cdc4. At least six such sites in Sic1 must be phosphorylated to exceed a physiological threshold for recognition (Price, 2002).

Loss of Sgg activity in wing disc clones induces Ci-155 to levels at least as high as the A/P border, but slightly lower than in PKA mutant clones. It is inferred that elevated Ci-155 levels result from inhibition of proteolysis to Ci-75 because ci RNA levels were unchanged and because Ci-75 repressor activity was largely absent from posterior smo sgg mutant clones in wing discs expressing Ci ubiquitously. In these clones there appears to be a low level of repressor, raising the possibility of a second GSK3 contributing in a minor way to the phosphorylation and proteolysis of Ci (Price, 2002).

Anterior sgg mutant clones induce some ectopic expression of Hh target genes but do not reproduce the strong phenotypes of PKA mutant clones, as assessed by Hh target gene expression, disc morphology, and adult morphology. Two possible explanations for the differences between PKA and sgg mutant clone phenotypes are offered. One possibility is that Ci lacking PKA site phosphorylation (and hence GSK3 and CK1 site phosphoserines) is more active than Ci lacking only GSK3 site phosphorylation. No significant differences have been found in the activity of Ci lacking three PKA sites (Ci3m), as compared to Ci lacking both GSK3 sites (CiNm) for the transgenic lines tested in wing discs or embryos. However, it was found that Ci lacking five consensus PKA sites (Ci5m) is more active than Ci3m. The fourth and fifth PKA sites are not flanked by GSK3 or CK1 sites. The phosphorylation of these PKA sites might therefore reduce the activity of Ci in sgg mutant clones relative to PKA mutant clones (Price, 2002).

A second possibility is that sgg mutations may prevent Hh target gene expression despite generating a phosphoform of Ci that is adequately activated and protected from proteolysis. This hypothesis was investigated by examining the phenotype of clones lacking both PKA and Sgg activities. Since GSK3 phosphorylation of Ci depends on priming by PKA phosphorylation, the absence of Sgg activity should not alter the phosphorylation state of Ci from that in PKA mutant clones. Nevertheless, the levels of Ptc protein induced in sgg PKA smo mutant clones are much lower than for PKA smo mutant clones in the wing pouch cells of the disc, and pattern duplication of wings normally associated with PKA mutant clones is suppressed by the additional inactivation of sgg. Thus, it is possible that a Sgg substrate in addition to Ci can affect Hh target gene expression, at least in anterior presumptive wing cells. This might contribute to the failure of sgg mutant clones in the wing pouch to induce ectopic Ptc expression (Price, 2002).

It is important to note that the positive input of Sgg on Hh target gene expression inferred above is evident only in the artificial circumstances of eliminating PKA and Sgg activities by genetic mutation. PKA and Sgg are normally active in anterior cells away from the A/P border, as manifested by the proteolysis of Ci-155, and any regulation of their activities by Hh at the A/P border is unlikely to be as dramatic as mutational inactivation. Indeed, there was no consistent reduction in the expression of ptc or dpp reporters in sgg mutant clones at the A/P border. Hence, the relevance of Sgg substrates other than Ci during normal development remains to be established (Price, 2002).

Shaggy/GSK3 antagonizes Hedgehog signaling by regulating Cubitus interruptus

The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate ortholog glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form(Ci75), and by stimulating the activity of accumulated Ci155. Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes including decapentaplegic and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signaling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3 phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. It is proposed that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets it for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation (Jia, 2002).

The proteolytic processing of Ci requires the activities of several intracellular Hh signaling components, including PKA and the kinesin-related protein Costal2 (Cos2). Overexpressing either Cos2 or a constitutively active form of PKA (mC*) blocks the accumulation of Ci155 induced by Hh. In contrast, wing discs overexpressing mC* or Cos2 accumulate high levels of Ci155 after treatment with 50 mMLiCl, a specific inhibitor of GSK3 kinase activity. These observations suggest that Sgg acts downstream of, or in parallel with, PKA and Cos2 to regulate Ci processing (Jia, 2002).

PKA promotes Ci processing by directly phosphorylating it at multiple sites in its carboxy-terminal region. Whether Sgg/GSK3 also regulates Ci processing by direct phosphorylation was also investigated. The canonical GSK3-phosphorylation site consists of two Ser/Thr residues separated by three amino acids: (Ser/Thr 0)-X-X-X-(Ser/Thr +4). Phosphorylation of Ser/Thr at the +4 position by a priming kinase allows GSK3 to phosphorylate Ser/Thr at the 0 position. Examination of Ci sequence reveals three GSK3 consensus sites (Ser 852, Ser 884 and Ser 888) adjacent to two previously identified PKA phosphorylation sites (Ser 856 and Ser 892). To determine whether Ci can be a direct substrate of Sgg/GSK3, an in vitro kinase assay was carried out. Three glutathione S-transferase (GST)-Ci fusion proteins containing Ci fragments from amino acids 441-1,065 were generated: GST-Ci contains wild-type sequence; GST-Ci-3P has three PKA sites mutated (S838A, S856A and S892A); and GST-Cim3 has three GSK3 consensus sites mutated (S852A, S884A and S888A). GST-Ci is specifically phosphorylated by PKA but not by GSK3 without prior PKA treatment; however, it becomes a good substrate for GSK3 after primed phosphorylation by PKA. In contrast, neither GST-Ci-3P nor GST-Cim3 can be phosphorylated by GSK3 even after PKA treatment. These results suggest that primed phosphorylation by PKA at Ser 856 and Ser 892 allows GSK3 to phosphorylate Ci at Ser 852, Ser 884 and Ser 888 (Jia, 2002).

Taken together, these data suggest that Sgg/GSK3 acts in conjunction with PKA to promote hyperphosphorylation of Ci, which is essential for efficient Ci processing to generate its repressor form. The requirement for multiple phosphorylation seems to be a general mechanism to regulate proteolysis of regulatory proteins. The involvement of multiple phosphorylation may provide a way for Hh to differentially regulate Ci by controlling its level of phosphorylation. For example, low levels of Hh may cause partial dephosphorylation of Ci by opposing Sgg, resulting in an inhibition of Ci processing to generate Ci75 but leaving Ci155 inactive. In contrast, high levels of Hh may cause complete dephosphorylation of Ci by opposing PKA, which not only blocks Ci processing but also stimulates the activity of accumulated Ci155 (Jia, 2002).

GSK3 is involved in multiple signaling pathways, raising the question of how its activity is selectively regulated by individual pathways. An emerging theme is that GSK3 is present, together with its substrates, in distinct complexes that are regulated by different upstream stimuli. Future study will determine whether Sgg/GSK3 forms a complex with Cos2 or Ci and whether Hh regulates Sgg/ GSK3 within the complex. In vertebrates, three Gli proteins (Gli1, Gli2 and Gli3) are implicated in transducing Hh signals. Interestingly, all three Gli proteins contain multiple GSK3-phosphorylation consensus sites adjacent to PKA sites, raising the possibility that GSK3 may regulate Gli proteins in vertebrate Hh pathways. Hh and Wnt signaling pathways act in synergy in certain developmental contexts. The finding that GSK3 is involved in both Hh and Wnt pathways raises the possibility that these two pathways might converge at GSK3 in certain developmental processes (Jia, 2002).

Modulation of Drosophila Slowpoke calcium-dependent potassium channel activity by bound Protein Kinase A catalytic subunit

Drosophila Slowpoke (Slo) calcium-dependent potassium channels bind directly to the catalytic subunit of cAMP-dependent protein kinase (PKAc). Coexpression of PKAc with Slo in mammalian cells results in a dramatic decrease of Slo channel activity. This modulation requires catalytically active PKAc but is not mediated by phosphorylation of S942, the only PKA consensus site in the Slo C-terminal domain. Slo binds to free PKAc but not to the PKA holoenzyme that includes regulatory subunits and is inactive. Activators of endogenous PKA that stimulate Slo phosphorylation, but do not produce detectable PKAc binding to Slo, do not modulate channel function. Furthermore, the catalytically inactive PKAc mutant does bind to dSlo but does not modulate channel activity. These results are consistent with the hypothesis that both binding of active PKAc to dSlo and phosphorylation of dSlo or some other protein are necessary for channel modulation (Zhou, 2002).

The initial evidence on modulation of calcium-dependent potassium channels by closely associated protein kinases or phosphatases came from experiments in which KCa channels reconstituted from rat brain were activated in an artificial lipid bilayer by the addition of ATP. PKAc was identified as one of several protein kinases closely associated with Slo channels. To determine whether the bound PKAc modulates channel function, Slo currents were recorded in the whole-cell voltage-clamp configuration. There is a dramatic downregulation of Slo channel functional activity with cotransfection of PKAc. Although it has been reported that channel function is not affected by coexpression with both PKAc and Src, it is now known that Src enhances Slo channel activity, and this upregulation by Src may have masked the PKAc downregulation that is described in this study (Zhou, 2002).

The reduction of whole-cell Slo in PKAc-cotransfected cells represents a change in channel functional activity. Although it is difficult to obtain an accurate conductance-voltage relation because of the large current amplitude in the whole-cell configuration, both the lower current amplitude and slower activation kinetics of Slo currents in PKAc-cotransfected cells are consistent with a decreased sensitivity of the Slo channel to voltage and calcium. The changes in current do not appear to result from alterations in channel expression or membrane targeting, because the expression level of Slo protein, measured by quantitative Western blot, is actually several-fold higher, and its surface localization measured by immunocytochemistry is unchanged, in PKAc-cotransfected cells. In any event, it is difficult to explain the change in channel kinetics in terms of protein expression level (Zhou, 2002).

Although questions still remain about the precise molecular mechanism underlying the profound modulation of Slo channel activity by cotransfected PKAc, the studies suggest strongly that it requires the association between active PKAc and the Slo channel. However, Slo channel activity is not altered by cotransfection of a catalytically inactive PKAc (K72E), although this mutant PKAc is capable of binding to Slo. This demonstrates that the modulation of Slo activity is not simply the result of binding of PKAc per se, but also requires phosphorylation. This is also consistent with other reports of modulation of native and expressed Slo family channels by PKAc-dependent phosphorylation. However, results with forskolin suggest that phosphorylation, although necessary, is not by itself sufficient to produce modulation. Accordingly, the hypothesis that modulation requires phosphorylation, of either Slo or some other protein, by active PKAc bound to the channel, is favored (Zhou, 2002).

To determine the potential molecular target the phosphorylation of which might mediate the modulation of Slo activity, the role of serine 942 in the Slo C-terminal domain was examined. This residue has long been recognized as a consensus PKA substrate, and it is readily phosphorylated by PKA both in vitro and in vivo. Interestingly, it was found that the mutation of serine 942 to alanine does not affect Slo binding to or modulation by PKAc. This is consistent with a previous report showing that S942A Slo is not different from the wild-type channel in its kinetic variability when expressed in Xenopus oocytes. Although it is convenient to use the anti-pS942 antibody to measure channel phosphorylation, this is not meant to imply that S942 participates in Slo modulation by PKAc (Zhou, 2002).

In addition to S942, other serine and threonine residues are thought to be exposed to the intracellular milieu. To identify other PKA substrate sites on Slo, an in vitro phosphorylation assay was performed using a recombinant fusion protein containing the entire Slo C-terminal domain. This domain is considerably longer than many other voltage-dependent potassium channels and constitutes approximately two-thirds of the total Slo channel protein. Somewhat surprisingly, it was found that other than S942, no additional amino acid in the entire Slo C-terminal domain is phosphorylated by PKAc in vitro. However, PKA phosphorylation of several serine or threonine residues in the intracellular loops between transmembrane domains has not been examined. It is equally plausible that PKAc phosphorylates other proteins that may themselves or through some signaling pathway modulate Slo channel activity. Slo is indeed regulated by several closely associated proteins. At least one Slo-interacting protein, 14-3-3, interacts with Slo via the adaptor protein Slob in a phosphorylation-dependent manner, and formation of this protein complex results in inhibition of channel activity. A recent report also showed that a Slo channel associating protein, cPLA2-alpha, is a target for phosphorylation. Phosphorylation of cPLA2-alpha results in activation of the channel, whereas phosphorylation of other regulatory elements causes channel inactivation. Finally, Slo channel activity is not modulated by activators of endogenous PKA, including forskolin and cBIMPS, which do not enhance binding of PKAc to Slo. Although these results do not lend themselves readily to unequivocal interpretation, they are consistent with the hypothesis that whatever the actual substrates of PKA are, targeting of sufficient PKAc via binding to Slo is also a requirement for channel modulation (Zhou, 2002).

It has been well documented that PKA forms regulatory complexes with some ion channels and ligand-gated receptors. PKA targeting is often achieved through AKAPs, proteins that bind to the PKA regulatory subunit as well as the substrate. However, overlay experiments showed a direct binding between Slo and PKAc, suggesting a novel channel-PKA protein complex. The present studies support this hypothesis and provide additional molecular details about this regulatory complex. Using co-immunoprecipitation approaches, it was shown that Slo binds only to free PKAc but not to the PKA holoenzyme, and that both PKA regulatory subunit and PKI inhibit the association between Slo and PKAc. A 35 amino acid region has been identified in the Slo C-terminal domain that is essential for PKAc binding. Interestingly, this region includes the consensus PKA substrate site, S942, although residues within the substrate site itself (RRXS) do not appear to be critical for binding to PKAc. This demonstrates that the Slo-PKAc association is not simply an enzyme-substrate complex. It is also consistent with previous studies on interactions between PKAc and regulatory subunits or PKI that show that residues separate from the pseudosubstrate site of the regulatory subunits or PKI are important in mediating the high-affinity binding to PKAc. Moreover, in vitro phosphorylation results suggest that binding of Slo to PKAc does not prevent the enzyme from phosphorylating its substrates. It remains to be determined how the activity of Slo-bound PKAc is regulated in cells (Zhou, 2002).

It is noteworthy that channels of the Slo family can physically associate with other protein kinases, including the Src tyrosine kinase and type I cGMP-dependent protein kinase. This suggests that modulation of channel activity may involve multiple regulatory mechanisms. Further biochemical and electrophysiological studies to dissect these regulatory pathways will undoubtedly provide important insights into the relationship between various signal transduction pathways and neuronal physiology (Zhou, 2002).

Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus

Hedgehog (Hh) proteins control animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh counteracts phosphorylation by PKA, GSK3, and CKI to prevent Cubitus interruptus (Ci) processing through unknown mechanisms. These kinases physically interact with the kinesin-like protein Costal2 (Cos2) to control Ci processing and Hh inhibits such interaction. Cos2 is required for Ci phosphorylation in vivo, and Cos2-immunocomplexes (Cos2IPs) phosphorylate Ci and contain PKA, GSK3, and CKI. By using a Kinesin-Cos2 chimeric protein that carries Cos2-interacting proteins to the microtubule plus end, it was demonstrated that these kinases bind Cos2 in intact cells. PKA, GSK3, and CKI directly bind the N- and C-terminal regions of Cos2, both of which are essential for Ci processing. Finally, it was shown that Hh signaling inhibits Cos2-kinase complex formation. It is proposed that Cos2 recruits multiple kinases to efficiently phosphorylate Ci and that Hh inhibits Ci phosphorylation by specifically interfering with kinase recruitment (Zhang, 2005).

To facilitate detection of protein-protein interaction between Cos2 and its binding partners in vivo, a Kinesin/Cos2 chimeric protein (Kinco) was generated in which the microtubule binding domain of Cos2 is replaced by the motor domain of Drosophila KHC. Kinco moves to the microtubule plus end and accumulates near the basal surface of imaginal disc epithelial cells. Strikingly, Kinco carries all the known Cos2 binding proteins to the same subcellular compartment, leading to colocalization. PKAc, GSK3, and two CKI isoforms, CKIα and CKIϵ, all colocalize with Kinco at the microtubule plus end, demonstrating that these kinases associate with Cos2 in intact cells. Hence, Kinco provides a powerful tool to determine if a protein interacts with Cos2 in vivo. In addition, Kinco colocalizes with Cos2-interacting proteins in cultured Drosophila cells such as S2 and cl8 cells. It is conceivable that one can use such a cell-based colocalization assay to identify additional proteins that form a complex with Cos2. Furthermore, it is also possible to extend this approach to other protein complexes by generating appropriate Kinesin chimeric 'bait' proteins (Zhang, 2005).

By using immunoprecipitation and GST pull-down assays, the kinase interaction domains were mapped to the microtubule binding (MB) and C-terminal (CT) of Cos2. GST fusion proteins containing either of these domains bind purified recombinant PKAc, GSK3, and CKI, suggesting that these kinases directly bind Cos2. However, the possibility cannot be rule out that these kinases may have additional contacts with other components in the Cos2 complex. Indeed, it was found that CKI can bind Ci in yeast (Zhang, 2005).

Several lines of evidence suggest that Cos2/kinase interaction plays an important role in regulating Ci phosphorylation and processing: (1) Ci phosphorylation is compromised in cos2 mutants; (2) the kinase-interacting domains in Cos2 are essential for Ci processing; (3) overexpressing multiple kinases can bypass the requirement of Cos2 for Ci processing (Zhang, 2005).

PKAc, GSK3, and CKI appear to bind competitively to Cos2; however, since Cos2 can dimerize and each Cos2 protein contains two kinase binding domains, a Cos2 dimer could in principle bind all three kinases simultaneously. It is possible that these kinases might not form a tight complex with Cos2 in a stoichiometric manner, which could explain why purification of endogenous Cos2 complexes failed to identify any of these kinases. However, by using in vitro kinase assay and Western blot analysis, the association of PKAc, GSK3, and CKI with endogenous Cos2 was detected. It is likely that interactions between Cos2 and kinases are transient; however, such interactions could increase local concentrations of these kinases; this greatly facilitates Ci hyperphosphorylation (Zhang, 2005).

It has been demonstrated that Hh induces Ci dephosphorylation in cl8 cells; however, it is not clear whether Hh blocks Ci phosphorylation by all three kinases or a subset of them. By using an antibody that specifically recognizes a phosphorylated PKA site in Ci, it was found that Hh partially inhibits PKA phosphorylation of Ci in wing discs. Consistent with this, Hh only partially blocks Cos2/PKA interaction. In contrast, Hh appears to have a more profound influence on the interaction between Cos2 and CKI or GSK3. Furthermore, CKI and GSK3 kinase activities associated with endogenous Cos2 diminishes upon Hh stimulation and Cos2IPs phosphorylates Ci to a lesser extent after Hh treatment. These observations suggest that Ci phosphorylation by CKI and GSK3 is likely to be inhibited by Hh in vivo (Zhang, 2005).

Several mechanisms may contribute to the regulation of Cos2-Ci-kinase complex formation by Hh. (1) The finding that PKAc, GSK3, and CKI bind Cos2 domains that also interact with Smo raises a possibility that Smo/Cos2 interaction may exclude kinases from binding to Cos2. Indeed, a membrane-tethered form of SmoCT (Myr-SmoCT) interferes with Cos2-Ci-kinase complex formation. (2) Smo/Cos2 interaction at the cell surface may induce conformational change in Cos2, which could mask its kinase interacting domains. (3) Cos2 is phosphorylated in response to Hh. Phosphorylation of Cos2 could regulate its interaction with one or more kinases. (4) There is evidence that Hh induces dissociation of Ci from Cos2, which may further decrease the accessibility of Ci to the kinases. This may explain why Hh induces more significant dissociation of PKAc from Ci than from Cos2. (5) Hh induces degradation of Cos2 in P compartment cells as well as in cells immediately adjacent to the A/P boundary; this may lead to a chronic destruction of Cos2-Ci-kinase complexes. However, it appears that only high levels of Hh induce Cos2 degradation in vivo. Low levels of Hh may prevent Ci phosphorylation with different mechanisms such as those described above (Zhang, 2005).

The following model is proposed for the regulation of Ci phosphorylation by Cos2 and Hh. In the absence of Hh, Cos2 scaffolds multiple kinases and Ci into proximity, thus increasing the accessibility of Ci to these kinases and facilitating extensive phosphorylation of Ci. Upon Hh stimulation, Cos2 complexes are recruited to the cell surface via Smo, leading to disassembly of Cos2-Ci-kinase complexes. As a consequence, Ci phosphorylation is compromised and Ci processing is blocked. This model has several interesting parallels to that proposed for the Wnt pathway. In quiescent cells, both pathways employ large protein complexes to bring kinases and their substrates in close proximity, resulting in phosphorylation and proteolysis of the transcription factor (Ci) or effector (β-catenin). Upon ligand stimulation, both pathways recruit the cytoplasmic signaling complex to the cell surface and cause dissociation of the complex, leading to dephosphorylation and stabilization of the transcription factor/effector. Interestingly, both pathways use common kinases, including GSK3 and CKI. However, these kinases together with their substrates form distinct signaling complexes assembled by pathway-specific scaffolding proteins (Cos2 and Axin in the Hh and Wnt pathways, respectively). Pathway activation is achieved by a specific interaction between the receptor system and the scaffolding protein (Smo/Cos2 interaction in the Hh pathway and LPR5/6/Axin interaction in the Wnt pathway). Thus, each pathway only controls the pool of kinases in the same complex with the pathway effector, leading to pathway-specific regulation of substrate phosphorylation. The combinatorial mechanism by which pathway-specific scaffolds bring common kinases into proximity with their substrates thus appears to be a general one and may apply to other signaling pathways that utilize a common set of kinases (Zhang, 2005).

Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb: Requirement for modification of Ci by PKA, CK1, and GSK3

Signaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb. Slimb is homologous to vertebrate ß-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis. Phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/ß-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, it is concluded that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. The phosphorylated motifs in Ci that are recognized by Slimb have been explored and some evidence is provided that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb (Smelkinson, 2006).

The mechanism and consequences of Hh signaling have been studied extensively in the developing Drosophila wing imaginal disc, where Hh, secreted from posterior compartment cells, induces a strip of nearby, responsive anterior cells (AP border cells) to express a small set of target genes, including decapentaplegic (dpp), that subsequently pattern the developing wing. In anterior cells far from Hh, Ci-155 is processed slowly to Ci-75, which crucially represses potential Hh target genes, including hh itself and dpp, to ensure that they are not ectopically expressed. Even low-level Hh signaling at the AP border blocks Ci-75 production, thereby also increasing the concentration of Ci-155. Hh further activates Ci-155 in a dose-dependent manner by facilitating its nuclear accumulation and potentially also by modifying its binding partners in the nucleus. Because formation of Ci-75 requires both Ci-155 phosphorylation and the activity of Slimb, it was proposed that Slimb might promote partial proteolysis of Ci-155 by directly binding to phosphorylated Ci-155 and catalyzing its ubiquitination. However, despite some support for this hypothesis, Ci-155 contains no obvious consensus binding site for Slimb/β-TRCP1: there are only two well-studied examples where proteasomal degradation of a ubiquitinated protein is incomplete (NF-KappaB precursors, p100 and p105), and ubiquitinated Ci-155 has not been detected when Ci-155 is stabilized by inhibiting the proteasome (Smelkinson, 2006).

To determine whether Slimb can bind to Ci in a manner dependent on phosphorylation, a purified GST fusion protein was used that includes the key phosphorylation sites of Ci. This GST-Ci protein undergoes a significant mobility shift in SDS polyacrylamide gels when phosphorylated by PKA and CK1 together and an even greater shift if GSK3 is also included. GST-Ci binds more avidly than GST alone to 35S-labeled full-length Slimb produced by in vitro translation in a reticulocyte lysate and to HA-tagged full-length Slimb from crude extracts of transiently transfected Drosophila Kc cells. This binding is reproducibly increased by using PKA together with CK1, and to a much greater extent by using all three protein kinases to phosphorylate GST-Ci prior to the binding assay. The synergistic contribution of GSK3 was clearest in the HA-Slimb binding assay, and so this assay was used to investigate further the characteristics of Ci binding to Slimb (Smelkinson, 2006).

Three PKA sites ('P1-3'), the neighboring three PKA-primed CK1 sites ('C1-3') and the two adjacent PKA-primed GSK3 sites ('G2,3') are required for Ci-155 to be converted to Ci-75 in Drosophila embryos and wing discs. When the serine residues at these PKA, CK1, or GSK3 sites were replaced with alanines, significant stimulation of binding of GST-Ci to HA-Slimb was no longer seen by any combination of PKA, CK1, and GSK3. Thus, strong binding of HA-Slimb to a Ci fragment in vitro requires the same protein kinases and the same phosphorylation sites that are required in vivo to convert Ci-155 to Ci-75 (Smelkinson, 2006).

Whether a defined, minimal Slimb binding site from another protein could direct processing of Ci-155 to Ci-75 was tested. β-catenin is a prototypical substrate for the β-TRCP1 SCF complex, in which a dually phosphorylated motif (DpSGIHpS, where pS stands for phosphoserine) is the critical recognition element for binding. This motif is conserved in Drosophila β-catenin (Armadillo), and Armadillo proteolysis depends on both this sequence and Slimb activity. The motif is also expected to serve as a direct binding site for Slimb, the Drosophila homolog of β-TRCP1. Tests revealed that this consensus Slimb/β-TRCP1 binding site, engineered into Ci, is functional and directs Slimb binding in vitro with an apparent avidity similar to that seen for fully phosphorylated wild-type Ci (Smelkinson, 2006).

Binding assays were used to search for the direct Slimb recognition elements in Ci because no established Slimb/β-TRCP consensus binding sites are apparent in the sequence of Ci or Gli proteins. Each of the three PKA sites in Ci is required for detectable processing to Ci-75 in Kc tissue-culture cells; therefore, whether each site contributes to Slimb binding in vitro was tested. Replacement of all three PKA-primed CK1 sites (and the two predicted CK1-primed CK1 sites) with acidic residues abolished any stimulation of HA-Slimb binding by phosphorylation of GST-Ci (Smelkinson, 2006).

It cannot be readily determined whether the PKA sites and PKA-primed CK1 sites are directly recognized by Slimb. However, the evident contribution of each PKA site to Slimb binding implies that each must nucleate at least one direct Slimb binding site. Surrounding the three PKA sites there are two types of motifs that are related to previously recognized or postulated Slimb/β-TRCP binding motifs (DSGXXS, DSGXXXS, TSGXXS, EEGXXS, DDGXXD, and DSGXXL. (1) The six-amino-acid motif (D/pS)(pS/pT)(Q/Y)XX(pS/pT) might be created in three places if phosphorylation occurs, as is suspected, at some nonconsensus sites. These motifs most closely resemble the β-TRCP1 binding site postulated for p100 (DpSAYGpS), but the presence of glutamine or tyrosine at the third position in place of glycine in the ideal consensus would be expected to reduce binding by more than an alanine substitution. (2) Many five-amino-acid motifs are created in which two acidic residues (DpS, pSpS, or pSpT) are separated from a phosphorylated residue (pS or pT) by only two amino acid residues. Four of these eight motifs include a glutamine at the third position. However, this residue does not appear to be instrumental in Slimb binding because substitution with alanine has little effect. On the basis of the crystal structure of β-TRCP1, it is hard to predict the affinity of the designated five-amino-acid motifs for Slimb, but it is likely to be lower than for any of the motifs cited above. Regardless of which precise motifs contribute most significantly to Slimb binding, it is clear from the mutational analysis that Ci must be highly phosphorylated over a region spanning almost sixty amino acid residues to generate several suboptimal binding sites that must collaborate to provide physiologically significant affinity for Slimb. This follows a precedent established for degradation of the yeast cell-cycle regulator Sic1 by binding of the SCFCDC4 complex to multiple low-affinity phosphodegrons. The requirement for extensive Ci phosphorylation could account for the essential role of the scaffolding protein Cos2 in facilitating phosphorylation and for the relatively slow conversion of Ci-155 to Ci-75 seen in vivo (Smelkinson, 2006).

In summary, processing of Ci-155 to Ci-75 is initiated by direct binding of Slimb to Ci-155 molecules that have been extensively phosphorylated by PKA, GSK3, and CK1. This presumably leads to ubiquitination of Ci-155 and its partial proteolysis by the proteasome, generating a transcriptional repressor that plays a key developmental role in cells that are not exposed to Hh. Whether phosphorylation also prevents Ci-155 from activating transcription through an additional mechanism remains to be explored, as does the mechanism by which proteolysis of Ci-155 is limited to preserve its N-terminal domains as Ci-75 (Smelkinson, 2006).

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. This study shows that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. When Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation (Smelkinson, 2007).

The central task of Hh signal transduction in Drosophila is to regulate the activity of the transcription factor Ci. This is accomplished by regulating the levels of Ci-155 activator and Ci-75 repressor and the specific activity of Ci-155. To examine the regulation of Ci-155-specific activity in isolation, a Ci variant (Ci-S849A) was developed that escapes PKA-dependent proteolysis but has a minimally altered pattern of PKA-initiated phosphorylation. At 29°C, Ci-S849A was expressed in wing discs (via C765-GAL4) at roughly the same level as endogenous Ci, but at 20°C, the levels of Ci-S849A were distinctly lower. At 29°C, Ci-S849A induced the Hh target gene reporter ptc-lacZ in A cells that are not stimulated by Hh. Since ptc induction requires Ci-155 activator and is not accomplished simply by loss of Ci-75 repressor, it is concluded that elevated levels of Ci-155 can suffice to confer some activator function. At 20°C, Ci-S849A induced ptc-lacZ in A cells only when Su(fu) was removed. By contrast, Ci-S849A activity was not detectably increased by loss of either PKA activity or Cos2 activity in P smo mutant clones, where Hh signaling is blocked. Thus, Su(fu) is the principal component that limits Ci-155-specific activity when Ci-155 is protected from PKA-dependent proteolysis, whereas PKA and Cos2 normally limit Ci-155 activity simply by promoting its proteolysisby promoting its proteolysis.

The prior assertion that PKA limits Ci-155-specific activity by direct phosphorylation was based on the use of an inappropriate reagent (Ci-U), which was mistakenly thought to be inert to PKA-dependent proteolysis. A similar role for Cos2 was previously inferred principally from the observation that Ci-155 accumulated more rapidly in the nuclei of anterior Leptomycin B-treated wing disc cells when those cells lack . It appears that the inferred cytoplasmic retention of Ci-155 by Cos2 contributes very little quantitatively to limiting the activity of stabilized Ci-155. That conclusion is supported by the observation that loss of Cos2 function did not enhance the weak induction of En seen in pka mutant clones of otherwise WT wing discs. The remaining, long-standing observations suggesting roles for PKA and Cos2 in limiting Ci-155-specific activity are the different degrees of Hh target gene induction in pka (strongest), cos2 (intermediate), and slimb (weakest) mutant wing disc clones. It is suggested that this might result from different degrees of disruption of PKA-dependent proteolysis in these clones. This suggestion is consistent with the proposed role of Cos2 in facilitating Ci-155 phosphorylation and with the observation that ptc-lacZ can be induced in slimb mutant clones when PKA activity is halved (Smelkinson, 2007).

Both GST-Ci association with SCFSlimb and Ci-155 proteolysis depend on two phosphorylated regions of Ci. The first region provides an essential Slimb binding site that can be created by five successive CK1 phosphorylations primed initially by PKA site 1. Of the four phosphorylated residues within the motif (844SpTpYYGSpMQSp852) that interacts directly with Slimb, at least one (S849) is essential for binding, and two others (S844 and S852) enhance binding (T845 is essential, but priming and binding functions have not been separated). The second critical phosphorylated region of Ci includes two GSK3 sites (S884 and S888) that are primed by PKA site 3. This region enhances, but is not sufficient for, binding to SCFSlimb (Smelkinson, 2007).

The requirement for multiple successive phosphorylations by PKA, CK1, and GSK3 to create a high-affinity SCFSlimb binding domain on Ci-155 has two important consequences. First, it demands a special mechanism for facilitating Ci phosphorylation that is met by Cos2. Second, it provides a mechanism through which a small change in Ci-155 phosphorylation, induced for example by limited dissociation of protein kinases from Cos2, can be translated into a substantial inhibition of Ci-155 proteolysis. The sharp increase in Ci-155 levels at the anterior limit of Hh signaling territory shows that a low dose of Hh does indeed severely curtail PKA-dependent Ci-155 proteolysis. Hh could inhibit proteolytic processing of Ci variants driven by either PKA-primed GSK3 sites (Ci-SL) or PKA-primed CK1 sites (Ci-G2,3E and Ci-Y846G), but complete inhibition was observed only for the latter pair. This suggests that the sensitive response of Ci proteolysis to Hh depends principally on CK1 (Smelkinson, 2007).

How does inhibition of Ci-155 proteolysis affect Ci-155 activity? Previously, the properties of Ci-U and slimb mutant clones were taken as evidence that inhibition of proteolysis does not suffice for Ci activation. Since Ci-U is subject to PKA-dependent proteolysis and PKA does not affect the specific activity of Ci-155, instead the properties of Ci-S849A and pka mutant clones were relied upon to conclude that complete inhibition of PKA-dependent proteolysis does suffice to induce the Hh target gene ptc. This, in turn, suggests that the high Ci-155 levels anterior to the stripe of elevated ptc expression at the AP border of wing discs result from substantial, but incomplete, inhibition of Ci-155 proteolysis (Smelkinson, 2007).

It has generally been assumed that inhibition of Ci-155 proteolysis is uniformly strong throughout the AP border and that the activation of ptc and en in nested domains is due solely to changes in Ci-155-specific activity elicited by increasing levels of Hh. However, several factors suggest that there may also be a significant gradient of residual PKA-dependent proteolysis at the AP border that contributes to morphogen action (Smelkinson, 2007).

First, the precise degree of substantially inhibited Ci-155 proteolysis can determine whether Hh target genes are induced or not. This is evident from differences in ptc-lacZ induction among proteolytically impaired Ci variants and between pka and slimb mutant clones (Smelkinson, 2007).

Second, Su(fu) is the principal regulator of Ci-155 activity when Ci-155 levels are elevated, yet Hh instructs an almost unchanged morphogenetic response in the absence of Su(fu). It is likely that a proteolytic gradient is critical under these conditions, although it is also possible that Cos2 assumes a more significant role in regulating Ci-155-specific activity when Su(fu) is absent (Smelkinson, 2007).

Third, it was found that the loss of any one of four phosphoserines that contribute to Ci-Slimb binding (S844, S852, S884, and S888) diminishes, but does not abolish, Slimb binding. For S888A (G3A) this results in elevated Ci-155 levels and an increased activity, but residual proteolysis is clearly evident from the generation of sufficient Ci-75 to repress hh-lacZ. Thus, dispersion of direct Slimb binding determinants among several phosphorylatable residues provides a mechanism for Hh to elicit graded inhibition of Ci-155 proteolysis. It is speculated that in response to high levels of Hh, most Ci-155 molecules will not bind to SCFSlimb at all because they lack at least two of the six key phosphorylated residues, whereas a large proportion of Ci-155 molecules may bind SCFSlimb with intermediate affinity in response to low or intermediate Hh levels because they lack only one critical phosphoserine (Smelkinson, 2007).

Fourth, regulation of Ci-155-specific activity depends on Ci-155 levels. Thus, Ci-155 is only activated by loss of Su(fu) when Ci-155 levels are elevated by Hh or appropriate mutations, presumably because other stoichiometric binding partners such as Cos2 act redundantly with Su(fu) when their Ci-155 binding capacity is not saturated. It would therefore be expected that the release of Ci-155 from repressive partners would be progressively facilitated as the relative levels of Ci-155 increase. This would allow increasing Hh levels to enhance Ci-155 activity through synergistic effects on Ci-155 levels and Ci-155-specific activity (Smelkinson, 2007).

The archetypal β-TRCP/Slimb substrates, β-catenin and IKB, contain a single, dually phosphorylated, high-affinity binding site (DSpGxxSp) that triggers rapid substrate proteolysis. The primary direct Slimb binding motif that has been defined (SpTpYYGSpMQSp) in Ci differs notably by the presence of Tyr instead of Gly at the third position, by the inclusion of a fourth electronegative residue at its C terminus, and by binding with lower affinity, permitting additional influences on Ci-SCFSlimb association. The fourth electronegative residue (pS852) likely interacts with at least one of two positively charged Slimb surface residues (R333 and R353) based on their potential proximity and reduced GST-Ci binding to the R333A/R353A Slimb variant (Smelkinson, 2007).

Most known β-TRCP substrates include phosphorylated or acidic residues that are two to four residues C-terminal to the standard six amino acid binding motif (DSpGXXSp), but their contribution to binding has not generally been assessed. Even β-catenin includes such phosphorylated residues that are known to have an essential priming role but have not been tested rigorously for direct interactions with β-TRCP. The variant β-TRCP binding motif (EEGFGSpSSp) of mammalian Wee1A presents a notable exception, in which a β-TRCP Arg residue equivalent to R353 of Slimb interacts with the phosphoserines at position 6 and 8 of this motif. This suggests that positive surface residues of β-TRCP/Slimb may commonly stabilize association with extended binding motifs. It is speculated that extended β-TRCP/Slimb binding motifs are likely to be especially important and prevalent in substrates lacking Gly at the third position because it was found that R333 or R353 (or both) of Slimb promotes binding to Ci-WT, but not to Ci-SL, and pS852 contributes significantly to Slimb binding in Ci-WT, but not in Ci-Y846G (Smelkinson, 2007).

Vertebrate Gli homologs of Ci also have a residue other than Gly at the third position (generally Ala) and a potentially phosphorylated Ser at the C terminus of a putative extended β-TRCP binding motif of 9 to 11 residues (SSAYx(x)SRRSS). Both a second Wee1A binding motif (DSAFQEPDS) and a β-TRCP binding motif of the p100 precursor of NFKB p52 (DSAYGSQSVE) also lack a Gly residue at the third position and include residues beyond the six amino acid core motif that might, by analogy to Ci, potentiate binding (Smelkinson, 2007).

Studies of Ci provide a clear precedent for the use of an extended β-TRCP/Slimb binding motif to translate regulated substrate phosphorylation into regulated proteolysis. However, it was also found that Slimb binding cannot be predicted by focusing on only the interactions of charged residues. Thus, fully phosphorylated Ci includes two sequences with a distribution of charged residues similar to that of the primary SCFSlimb binding site (837DSpQNSpTpASpTp and 858SpSpQVSpSpIPTp compared with 844SpTpYYGSpMQSp), but those sites neither suffice for Slimb binding (in Ci-S849A) nor enhance binding significantly in vitro (as revealed by D837A, T842A, S858A, S859A, and Ds2 variants). This probably reflects significant binding contributions of nonpolar residues in positions 3-5 of an extended Slimb/β-TRCP binding motif, as suggested by the presence of Tyr or Phe at position 4 of the functional motifs of Ci, Gli, p100, and Wee1A (Smelkinson, 2007).

Several F-box proteins that use WD40 repeats to bind substrate (FBW proteins) also include a dimerization domain that directs assembly of higher-order SCF complexes. Some substrates of these SCF complexes (for example, Cyclin E for Fbw7 and Wee1A for β-TRCP) contain more than one phosphorylated region capable of interacting with the same WD40 binding surface of the FBW protein. This raises the question of whether the cooperative function of two or more such regions depends on SCF dimerization and simultaneous interaction with two FBW subunits of a dimeric complex (Smelkinson, 2007).

This study found that Ci also contains two phosphorylated regions that contribute to SCF association, and Slimb molecules can bind to each other within higher-order functional SCF complexes. It was also found that Slimb self-association enhanced binding to GST-Ci relative to GST-Ci-SL, which contains a single DSGxxS motif; however, it was not required for both phosphorylated regions of GST-Ci to stimulate binding. Hence, simultaneous binding to separate Slimb monomers within a larger complex can be excluded as a requisite mechanism for cooperativity between the two phosphorylated regions of Ci. This is consistent with recent structural studies that predict a wider separation of WD40 binding surfaces within an SCF dimer than can be spanned readily by the two critical phosphorylated regions of a single Ci molecule (Smelkinson, 2007).

Does the region preceding PKA site 3 of Ci bind directly to the FBW component (Slimb) of an SCF complex as for Cyclin E and Wee1A? That model was proposed for Gli-2/3 proteins, which include a recognizable, potentially extended, variant Slimb-binding motif (DSYDPISTDAS). The analogously positioned sequence in Drosophila (SFYDPISPGCS) retains the YDPIS sequence and the two GSK3 sites at position 7 and 11 (underlined) but lacks a Ser at position 2 (italics), which is required in Gli-2/3 for normal β-TRCP association and proteolysis. Also, Ala substitution of the first Ser in the Drosophila motif (together with three other Ser residues) had only a minor effect on Slimb binding in vitro and Ci-155 proteolysis in vivo. Thus, this region of Ci does not have a clearly recognizable and demonstrably functional, conventional β-TRCP/Slimb binding site. It is, nevertheless, conceivable that the conserved elements of the putative β-TRCP binding motif of Gli-2/3 might provide a very weak direct interaction with the WD40 domain of Slimb that is sufficient to enhance SCFSlimb association (Smelkinson, 2007).

However, this study also found that the GSK3 enhancement of Ci-Slimb binding conferred by the GSK3 sites preceding PKA site 3 was lost if Slimb lacked an F-box domain and consequent direct association with SkpA and its SCF complex partners. This result is interpreted with some caution because Slimb-ΔF also bound less well than wild-type Slimb to a canonical β-catenin substrate and to GST-Ci that was phosphorylated only at its primary Slimb binding site. Nevertheless, the result suggests that the region of Ci immediately preceding PKA site 3 might augment SCF association by binding directly to an SCF component other than Slimb (Smelkinson, 2007).

Whether GSK3 stimulates Ci binding to SCFSlimb via a direct interaction with Slimb, an unprecedented interaction with another SCF component, or a conformational effect on the primary Slimb binding site of Ci, the Ci-G3A transgene reveals that the stimulation conferred by GSK3 phosphorylation is critical for efficient Ci-155 proteolysis and for Hh pathway silencing. Since Slimb self-association enhanced GST-Ci, but not GST-Ci-SL, binding in vitro, it is suspected that this may also be important for Ci-155 proteolysis in vivo. It is not known if SCFSlimb dimerization (or oligomerization) is regulated, but the different modes of association of SCFSlimb with Ci and β-catenin certainly provide several opportunities for SCF regulatory mechanisms or mutations to affect the Hh pathway without altering the Wnt/β-catenin pathway (Smelkinson, 2007).

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

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

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

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

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

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

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

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

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

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

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

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

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

Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation

Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. This study shows that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. Direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and evidence is provided for an analogous action of PKA on Ci (Marks, 2011).

There are clearly many conserved features of Hh signaling between Drosophila and mammals, and some explicit differences, most obviously with regard to the employment of cilia. However, incomplete understanding can lead to premature conclusions as to exactly what is conserved. When studies of Gli activity were initiated in Drosophila, the absence of key fly Smo activation and Cos2-binding domains from mammalian Smo, coupled to studies showing no effect of Kif7 and Kif27 RNAi on the Hh pathway in tissue culture cells, had led to the suggestion that a Cos2-like molecule was not important in regulating Gli activity in mammals. This study has shown that Cos2 is essential for Gli3 processing into a repressor form in Drosophila, that Cos2 prevents constitutive Gli1 activity while permitting Gli1 activation by Hh in Drosophila and that Cos2 binds to three distinct regions of Gli1, as observed for Ci. These results strongly indicated that a Cos2-like molecule must regulate the activity of Gli proteins in mammals. Indeed, genetic elimination of Kif7 in mouse was subsequently found to reduce processing of Gli3 and was also inferred to result in partial activation of Gli2 in the absence of a Hh ligand. The fundamental demonstration of conservation of Cos2 function focuses attention on more precise questions about how Cos2-family proteins regulate Ci/Gli protein function. Some of these questions have been addressed by comparing the regulation of Ci and Gli proteins in Drosophila (Marks, 2011).

Although physiological regulation of Gli1 protein activity by Shh was demonstrated by knocking Gli1 coding sequence into the mouse Gli2 locus, those mice have not been extensively characterized further to test which specific factors regulate Gli1 activity in mouse. In mice with wild-type Gli genes, the dependence of Gli1 transcription on Hh pathway activity coupled with the ubiquitous presence of Gli2 as a major sensor of pathway activity has prevented an assessment of post-transcriptional regulation of Gli1. For example, genetic loss of mouse suppressor of fused (Sufu) leads to substantial activation of Shh-target genes and transcriptional induction of Gli1, leading only to the conclusion that Sufu normally limits Gli2 activity. Similarly, the effects of mutations affecting cilia or Kif7 have been interpreted only with respect to Gli3 and Gli2 activities (Marks, 2011).

This study found that silencing Gli1 in the absence of Hh signaling requires PKA, PKA sites S544 and S560, Cos2 and the Cos2-binding region equivalent to CORD in Ci, but that T374 and the DSGVEM motif, which has been previously implicated in Gli1 regulation, are not required for Gli1 silencing or activation by Hh. Although the known dependence of Ci phosphorylation on Cos2 suggests that Cos2 might be required to promote Gli1 phosphorylation by PKA, the greater activity of Gli1 lacking the CORD region compared with Gli1 lacking PKA sites in anterior wing disc cells suggests that Cos2 may do more than simply promote Gli1 phosphorylation (Marks, 2011).

How is Gli1 silenced by PKA phosphorylation in Drosophila? Ci silencing involves equivalent PKA sites (in position and immediate sequence context) and creates a Slimb-binding site essential for Ci-155 processing. However, additional PKA and PKA-primed phosphorylation sites important for efficient Ci and Gli3 proteolysis are absent from Gli1. Thus, it would be difficult to predict a priori whether PKA and Hh alter Gli1 activity by regulating its proteolysis (Marks, 2011).

Direct assessment of Gli1 protein levels (using a C-terminal HA tag) in wing disc cells indicated that neither Hh stimulation nor PKA inhibition increased Gli1 levels. However, Ci-155 levels can be a deceptive indicator of PKA-dependent proteolysis because Ci-155 can also be degraded by an activation-dependent mechanism that is independent of PKA. Hence, Ci-155 levels are elevated at intermediate levels of Hh signaling, but in cells exposed to the highest levels of Hh, where PKA-dependent processing is strongly inhibited, Ci-155 levels are very similar to those in anterior cells with no Hh exposure. The observation that Gli1 levels may actually be slightly reduced in posterior cells relative to anterior cells, and in anterior clones deficient for PKA, suggests that activation may also de-stabilize Gli1 and could disguise any putative, opposite contribution from PKA-dependent proteolysis. Consequently, evidence of PKA-dependent proteolysis was sought by creating a Ci-Gli1 fusion protein for which PKA-dependent proteasome digestion would be expected to be arrested and produce Ci-75 repressor. The activity of the Ci-Gli1 fusion protein was regulated by PKA, Cos2 and Hh, as for Gli1 and Ci, but no repressor was formed. Hence, no evidence was found of PKA-dependent proteolysis of Gli1. Weak induction of ptc-lacZ expression in anterior cells was found to require much higher levels of Gli1 protein than evident for equivalent (or greater) ptc-lacZ induction by Gli1 lacking PKA sites 544 and 560, or by wild-type Gli1 in response to Hh. Thus, both PKA inhibition and Hh clearly increase the specific activity of Gli1 and do not appear to stabilize Gli1 protein (Marks, 2011). If PKA principally regulates the specific activity of Gli1 in Drosophila, it might be expected that the same mechanism can regulate Ci-155 activation. Direct evidence for such a role was not observed previously when comparing the activity of low levels of proteolysis-resistant Ci (Ci-S849A) in posterior smo and smo pka mutant clones, even though it is clear that endogenous Ci-155 is activated more strongly by loss of PKA than by loss of Slimb. Likewise, loss of Cos2 activates Ci-155 more strongly than loss of Slimb but did not detectably increase the activity of Ci S849A in posterior smo mutant clones. Su(fu) also has the potential to limit Ci-155 activity. By eliminating Su(fu), this study revealed a repressive influence of both PKA and Cos2 on Ci-S849A in posterior smo mutant clones. It is concluded that the potentially redundant effects of PKA, Cos2 and Su(fu) limiting Ci-155 activity can each be exposed in the absence of Ci-155 processing but are probably most readily seen at high Ci-155 levels. The experiments with Ci do not reveal the mechanism by which PKA and Cos2 can limit Ci-155 activity. An equivalent regulatory influence was much clearer for Gli1; it involves direct Cos2 binding and phosphorylation of defined PKA sites, providing an important starting point for further investigation (Marks, 2011).

Kif7 is known to promote the proteolysis of Gli2 and Gli3 in mouse. This study has shown that Kif7 can also promote Ci-155 and Gli3 processing in Drosophila. Cos2 promotes Gli3 processing and limits Gli1 activity in Drosophila. In all of these cases, PKA and conserved PKA sites have analogous effects to Cos2, consistent with the common hypothesis that Cos2 and Kif7 can promote phosphorylation of Ci-155 and Gli proteins at specific PKA and PKA-primed sites (Marks, 2011).

This study has found that Cos2 effects on Gli1 required the CORD-equivalent region of Gli1. Regulation of Ci-155 by Cos2 does not require the CORD region of Ci. In fact, Ci-155 can be processed and silenced in the absence of both CDN and CORD domains, provided a third Cos2 interaction domain within its zinc fingers is present. Gli1 shares all three Cos2 interaction domains. Moreover, Kif7 can promote processing of Ci lacking CDN and CORD regions, implying that a single Ci/Gli interface with a Cos2/Kif7 protein can silence Ci-155 but not Gli1. It is possible that a single Cos2/Ci-155 interface suffices because it is stabilized by additional common binding partners, Fu and Su(fu), and that these proteins do not contribute efficiently to Cos2/Gli1 binding (Marks, 2011).

Most importantly, Hh inhibits the Cos2-dependent processing of Gli3 and Cos2-dependent silencing of Gli1 in Drosophila. The mechanisms by which Hh opposes Cos2 actions on Ci-155 are not yet fully resolved. One potential mechanism, the dissociation of PKA, CK1 and GSK3 from Cos2, would be expected to influence both Ci and Gli protein activities. However, there is probably also a role for Hh-dependent dissociation of Cos2 from Ci, implying that the interactions between Cos2 (or Kif7) and Gli proteins may be regulated by an analogous mechanism (Marks, 2011).

Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity

Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. This study shows that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity (Cho, 2015).

These findings indicate that the SNARE-binding protein Cpx is a key PKA target that regulates spontaneous fusion rates and presynaptic plasticity at Drosophila NMJs. Cpx's function can be modified to regulate activity-dependent functional and structural plasticity. In vivo experiments using Cpx phosphomimetic rescues demonstrate that Cpx phosphorylation at residue S126 selectively alters its ability to act as a synaptic vesicle fusion clamp. In addition, S126 is critical for the expression of HFMR, an activity-dependent form of acute functional plasticity that modulates mini frequency at Drosophila synapses. These data indicate a Syt 4-dependent retrograde signaling pathway converges on Cpx to regulate its synaptic function. Additionally, it was found that elevated spontaneous fusion rates correlate with enhanced synaptic growth. This pathway requires Syt 4 retrograde signaling to enhance spontaneous release and to trigger synaptic growth. Moreover, the Cpx S126 PKA phosphorylation site is required for activity-dependent synaptic growth, suggesting acute regulation of minis via Cpx phosphorylation is likely to contribute to structural synaptic plasticity. Together, these data identify a novel mechanism of acute synaptic plasticity that impinges directly on the presynaptic release machinery to regulate spontaneous release rates and synaptic maturation (Cho, 2015).

How does acute phosphorylation of S126 alter Cpx's function? The Cpx C-terminus associates with lipid membranes through a prenylation domain (CAAX motif) and/or the presence of an amphipathic helix. The Drosophila Cpx isoform used in this study (Cpx 7B) lacks a CAAX-motif, but contains a C-terminal amphipathic helix flanked by the S126 phosphorylation site. S126 phosphorylation does not alter synaptic targeting of Cpx or its ability to associate with SNARE complexes in vitro. As such, phosphorylation may instead alter interactions of the amphipathic helix region with lipid membranes and/or alter Cpx interactions with other proteins that modulate synaptic release. Given the well-established role of the Cpx C-terminus in regulating membrane binding and synaptic vesicle tethering of Cpx, phosphorylation at this site would be predicted to alter the subcellular localization of the protein and its accessibility to the SNARE complex. However, no large differences between WT Cpx and CpxS126D were observed in liposome binding. This assay is unlikely to reveal subtle changes in lipid interactions by Cpx, as this study found that C-terminal deletions (CTD) maintained its ability to bind membranes. The ability of the CTD versions of Drosophila Cpx to associate with liposomes is unlike that observed with C. elegans Cpx, and indicate domains outside of Drosophila Cpx's C-terminus contribute to lipid membrane association as well, potentially masking effects from S126 phosphorylation that might occur in vivo. Alternatively, phosphorylation of the Cpx C-terminus could alter its association with other SNARE complex modulators such as Syt 1 (Cho, 2015).

The data indicate that enhanced minis regulate synaptic growth through several previously identified NMJ maturation pathways. The Wit signaling pathway is required for synaptic growth in the background of enhanced minis. The wit gene encodes a presynaptic type II BMP receptor that receives retrograde, transsynaptic BMP signals from postsynaptic muscles. Consistent with these data, other studies have demonstrated that downstream signaling components of the BMP pathway are necessary and sufficient for mini-dependent synaptic growth at the Drosophila NMJ. Additionally, it was found that the postsynaptic Ca++ sensor, Syt 4, is required for enhancing spontaneous release and increasing synaptic growth. The data does not currently distinguish the interdependence of the BMP and Syt 4 retrograde signaling pathways, and other retrograde signaling pathways might contribute to mini-dependent synaptic growth as well. Recently, several retrograde pathways have been identified that regulate functional homeostatic plasticity at the Drosophila NMJ. Future work will be required to fully define the retrograde signaling pathways necessary to mediate mini-dependent synaptic growth (Cho, 2015).

How might elevated spontaneous release through Cpx phosphorylation regulate synaptic growth? It is hypothesized that the switch in synaptic vesicle release mode to a constitutive fusion pathway that occurs over several minutes following stimulation serves as a synaptic tagging mechanism. By continuing to activate postsynaptic glutamate receptors in the absence of incoming action potentials, the elevation in mini frequency would serve to enhance postsynaptic calcium levels by prolonging glutamate receptor stimulation. This would prolong retrograde signaling that initiates downstream cascades to directly alter cytoskeletal architecture required for synaptic bouton budding. Given that elevated rates of spontaneous fusion still occur in cpx and syx3-69 in the absence of Syt 4 and BMP signaling, yet synaptic overgrowth is suppressed in these conditions, it is unlikely that spontaneous fusion itself directly drives synaptic growth. Rather, the transient enhancement in spontaneous release may serve to prolong postsynaptic calcium signals that engage distinct effectors for structural remodeling that fail to be activated in the absence of elevated spontaneous release. Results from mammalian studies indicate spontaneous release can uniquely regulate postsynaptic protein translation and activate distinct populations of NMDA receptors compared to evoked release, so it is possible that spontaneous fusion may engage unique postsynaptic effectors at Drosophila NMJs as well (Cho, 2015).

In the last few decades, intense research efforts have elucidated several molecular mechanisms of classic Hebbian forms of synaptic plasticity that include long-term potentiation (LTP) and long-term depression (LTD), alterations in synaptic function that lasts last minutes to hours. The most widely studied expression mechanism for these forms of synaptic plasticity involve modulation of postsynaptic AMPA-type glutamate receptor (AMPAR) function and membrane trafficking. In contrast, molecular mechanisms of short-term synaptic plasticity remain poorly understood. Several forms of short-term plasticity have been described, such as post-tetanic potentiation (PTP), which involves stimulation-dependent increases in synaptic strength, including changes in mini frequency. Short-term plasticity expression is likely to impinge on the alterations to the presynaptic release machinery downstream of activated effector molecules. For example, Munc 18, a presynaptic protein involved in the priming step of vesicle exocytosis via its ability to associate with members of the SNARE machinery, is dynamically regulated by Ca++-dependent protein kinase C (PKC), and its regulation is required to express PTP at the Calyx of Held. This study demonstrates that the presynaptic vesicle fusion machinery can also be directly modified to alter spontaneous neurotransmission via activity-dependent modification of Cpx function by PKA. Protein kinase CK2 and PKC phosphorylation sites within the C-terminus of mammalian and C. elegans Cpx have been identified. Therefore, activity-dependent regulation of Cpx function via C-terminal phosphorylation may be an evolutionarily conserved mechanism to regulate synaptic plasticity. Moreover, Cpx may lie downstream of multiple effector pathways to modulate various forms of short-term plasticity, including PTP, in a synapse-specific manner. Interestingly, Cpx is expressed both pre- and postsynaptically in mammalian hippocampal neurons and is required to express LTP via regulation of AMPAR delivery to the synapse, suggesting Cpx-mediated synaptic plasticity expression mechanisms may also occur postsynaptically (ACho, 2015 and references therein).

In summary, these results indicate minis serve as an independent and regulated neuronal signaling pathway that contributes to activity-dependent synaptic growth. Previous studies found Cpx’s function as a facilitator and clamp for synaptic vesicle fusion is genetically separable, demonstrating distinct molecular mechanisms regulate evoked and spontaneous release. Evoked and spontaneous release are also separable beyond Cpx regulation, as other studies have demonstrated that minis can utilize distinct components of the SNARE machinery, distinct vesicle pools, and distinct individual synaptic release sites . These findings suggest diverse regulatory mechanisms for spontaneous release that might be selectively modulated at specific synapses (Cho, 2015).

Continued: see cAMP-dependent protein kinase 1: Protein Interactions part 2/2

cAMP-dependent protein kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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