The Contributions of Protein Kinase A and Smoothened Phosphorylation to Hedgehog Signal Transduction in Drosophila melanogaster

Abstract

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. Here we show 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, we show that Smo containing acidic residues at PKA and CK1 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.

THE Hedgehog (Hh) signaling pathway regulates many aspects of cell specification and cell proliferation in organisms from Drosophila to humans and is of prime medical importance, especially because its genetic alteration contributes to several forms of cancer (Ingham and McMahon 2001; McMahon et al. 2003; Pasca di Magliano and Hebrok 2003; Yamada et al. 2004; Hooper and Scott 2005). Although there are some indications of significant differences in the mechanism of Hh signal transduction between Drosophila and vertebrate cells (Huangfu and Anderson 2006; Svard et al. 2006; Varjosalo et al. 2006), there is also clearly a great deal of conservation, and the bulk of our current understanding derives from studies in Drosophila (Hooper and Scott 2005). Key conserved components include the Hh receptor Patched (Ptc), the transmembrane protein Smoothened (Smo), and the transcriptional effector Cubitus interruptus (Ci; homologous to Gli proteins in vertebrates). In all cases Ptc silences the pathway in the absence of ligand by allowing processing of Ci or Gli proteins to forms that act as transcriptional repressors, while limiting the accumulation and activity of full-length Ci or Gli transcriptional activators. When Hh family ligands bind to Ptc the processing of Ci/Gli proteins is blocked and these proteins instead become effective transcriptional activators. Smo is required to mediate these responses to Hh ligands but neither the regulation of Smo activity by Ptc and Hh nor the effector functions of Smo are well understood (Hooper and Scott 2005).

Protein phosphorylation is instrumental in both the silencing and the activation of the Hh pathway. Proteolytic conversion of full-length Drosophila Ci (Ci-155) to the Ci-75 repressor requires phosphorylation of Ci-155 by protein kinase A (PKA) at three defined sites, followed by phosphorylation at neighboring PKA-primed casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3) sites (Aza-Blanc et al. 1997; Jia et al. 2002; Price and Kalderon 2002). Phosphorylation at these and further primed sites by these three protein kinases creates a binding site for the Skp1/Cullin1/F-box component Slimb (Jia et al. 2005; Smelkinson and Kalderon 2006). This presumably leads to Ci-155 ubiquitination and partial proteolysis by the proteasome (Maniatis 1999; Tian et al. 2005). Vertebrate Gli proteins include analogous PKA, CK1, and GSK3 sites and the proteolysis of Gli3 and Gli2 depends on these sites and the consequent binding of the Slimb homolog β-TRCP (Pan et al. 2006; Wang and Li 2006). Furthermore, Hh signaling can regulate the partial proteolysis of Gli3 protein, both in vertebrates and when Gli3 is introduced into Drosophila (von Mering and Basler 1999; Aza-Blanc et al. 2000; Wang et al. 2000). In addition to promoting repressor formation, phosphorylation may also limit the specific activity of Ci-155 as a transcriptional activator (Wang et al. 1999). Through these actions on Ci/Gli proteins PKA silences the Hh pathway in Drosophila and vertebrates in the absence of ligand.

Phosphorylation also affects Smo activity. Drosophila Smo requires a cluster of three PKA sites and the adjacent PKA-primed CK1 sites in its sizable carboxy-terminal cytoplasmic domain to transduce an Hh signal (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). Accordingly, Hh pathway activity can be reduced by inhibiting PKA and CK1 activities but this deficit in signaling is hard to measure and evaluate accurately because loss of PKA or CK1 simultaneously contributes to activation of Ci-155 independent of any input from Hh or Smo (Jia et al. 2004; Apionishev et al. 2005). Alteration of all of the clustered PKA and adjacent primed CK1 sites in Smo to acidic residues, potentially mimicking phosphorylation, confers some Hh-independent activity on Smo (Jia et al. 2004; Zhang et al. 2004). Hence, Smo phosphorylation on PKA and CK1 sites is necessary for activity and may even suffice to activate Smo. These critical PKA and CK1 sites are not conserved in vertebrate Smo proteins, which have smaller carboxy-terminal cytoplasmic domains, implying a notable difference in the design of vertebrate and invertebrate Hh pathways (Huangfu and Anderson 2006; Varjosalo et al. 2006). There is, however, some evidence that Hh-dependent phosphorylation of vertebrate Smo at G-protein receptor kinase sites is required for activity, potentially reflecting a parallel regulatory mechanism (Chen et al. 2004; Wilbanks et al. 2004; Kalderon 2005).

While the cited evidence clearly points to a role for phosphorylation of Smo at defined PKA and CK1 sites in Hh pathway activation there are a number of apparent inconsistencies in the data supporting this idea, including the markedly different potencies of excess PKA activity in inducing Hh target genes in embryos compared to wing discs (Ohlmeyer and Kalderon 1997; Jia et al. 2004). Here we resolve some of these inconsistencies. In doing so, we find that strong induction of Hh target genes in embryos by excess PKA activity depends on regulatory elements other than Ci binding sites and we explore further how Smo phosphorylation affects Hh pathway activity.

MATERIALS AND METHODS

Immunohistochemistry and RNA in situ hybridization:

Embryo in situ hybridization was performed as described previously (Ohlmeyer and Kalderon 1997) using digoxigenin-labeled RNA probes for wg, ptc, and E. coli lacZ gene products described previously (Ohlmeyer and Kalderon 1997; Lessing and Nusse 1998). Third instar wing discs were stained as in Smelkinson and Kalderon (2006) with rabbit anti-β-galactosidase and mouse anti-En monoclonal 4D9 (DSHB), using AlexaFluor594 and AlexaFluor488 secondaries, respectively, or AlexaFluor647 for En staining when GFP was also present to mark mutant clones.

Crosses for embryo assays:

Consequences of ectopic expression of active mouse PKA catalytic subunit (mC*), PKA inhibitor (R*) (Ohlmeyer and Kalderon 1997), or Hh on the wg gene reporters wg-lacZ5.1, WLZGc2.5L (Lessing and Nusse 1998), Δwg-lacZ, Δwg*-lacZ (Von Ohlen and Hooper 1997), the ptc gene reporters ptc-lacZ, FE-lacZ (Forbes et al. 1993; Alexandre et al. 1996), and the Ci binding site reporter Ci-Grh-lacZ (Barolo and Posakony 2002) were assayed in the following crosses (where RG1 and ptc-GAL4 are enhancer trap insertions of a GAL4 transgene; Ohlmeyer and Kalderon 1997):

1. RG1 (= prd-GAL4)/TM3 crossed to UAS-mC*; wg-lacz5.1, to UAS-Hh wg-lacZ5.1, to UAS-mC*; Ci-Grh-lacZ/TM6B, to UAS-R*; Ci-Grh-lacZ, to UAS-Ci-H5m-w1; Ci-Grh-lacz/TM6B, to UAS-mC* UAS-Ci-H5m-w1; Ci-Grh-lacZ/TM6B, or to UAS-R* UAS-Ci-H5m-w1; Ci-Grh-lacZ/TM6B.

2. WLZGc2.5L; RG1/TM3 or Δwg-lacZ; RG1/TM3 or Δwg*-lacZ; RG1/TM3 crossed to UAS-mC* or to UAS-Ci-T5m-s1.

3. ptc-GAL4; Ci-Grh-lacZ/TM6B or ptc-GAL4; FE-lacZ or ptc-GAL4; ptc-lacZ crossed to UAS-mC* or to UAS-Hh.

4. ptc-GAL4; Su(fu)^LP Ci-Grh-lacZ/TM6B crossed to Su(fu)^LP or to UAS-mC*; Su(fu)^LP.

Wg requirements for wg and ptc reporter expression in embryos were assayed in wg^cx4 ptc^S2/CyO ftz-lacZ; wg-lacZ5.1 and wg^cx4 ptc^S2/CyO ftz-lacZ; FE-lacZ/TM2 stocks and in crosses of wg^cx4 UAS-mC*/CyO; wg-lacZ5.1 to wg^cx4/CyO; RG1/TM2 stocks.

SmoD1-3 was expressed in alternating segments of smo mutant embryos, together with Hh or mC* in crosses of y hs-flp/yw; smo² ck FRT40A/ P[ovo]^D FRT40A; RG1/+ females that were heat-shocked for 1 hr at 37° as third instar larvae to males that were smo² FRT40A/CyO; UAS-SmoD1-3/TM6B or smo² FRT40A UAS-mC*/CyO; UAS-SmoD1-3/TM6B or smo² FRT40A/CyO; UAS-SmoD1-3 UAS-Hh/TM6B. Embryos homozygous for smo² (and lacking germline smo activity) were readily identified as lacking wg and ptc expression in segments that do not express prd-GAL4 (RG1).

Crosses for wing disc assays:

C765-GAL4 ptc-lacZ/TM6B or Su(fu)^LP C765-GAL4 ptc-lacZ/TM6B flies were crossed to UAS-mC*, to UAS-mC*; UAS-dbt, to UAS-Ci-H5m-w1, to UAS-Ci-H5m-w1 UAS-mC*, to UAS-mC*; Su(fu)^LP, to UAS-Ci-H5m-w1; Su(fu)^LP, or to UAS-Ci-H5m-w1 UAS-mC*; Su(fu)^LP flies. Possible rescue of smo and PKA-C1 mutant clone phenotypes by SmoD1-3 was tested in animals of the following genotypes heat-shocked at second instar to induce homozygous mutant clones: yw hs-flp/yw; smo² ck FRT40A/Ubi-GFP FRT40A; C765-GAL4 ptc-lacZ/UAS-SmoD1-3, yw hs-flp/yw; smo² ck PKA-C1^B3 FRT40A/Ubi-GFP FRT40A; C765-GAL4 ptc-lacZ/UAS-SmoD1-3, and yw hs-flp/yw; ck PKA-C1^B3 FRT40A/Ubi-GFP FRT40A; C765-GAL4 ptc-lacZ/+. Discs expressing SmoD1-3 but lacking Fu kinase activity were dissected from y male larvae from the cross of yw fu^mH63/yw fu^mH63; P[y⁺] P[Fu⁺]/CyO; C765-GAL4 ptc-lacZ/+ to UAS-SmoD1-3. Fu phosphorylation was assayed as in Apionishev et al. (2005) in extracts prepared as in Smelkinson and Kalderon (2006) of discs from C765-GAL4 ptc-lacZ/+, UAS-mC*/+; C765-GAL4 ptc-lacZ/+, UAS-mC*/+; C765-GAL4 ptc-lacZ/ UAS-dbt, or C765-GAL4 ptc-lacZ/UAS-SmoD1-3 larvae raised at 29°.

RESULTS

Can excess PKA activity induce Hh pathway activity in wing discs?

Conversion of PKA and CK1 sites on Smo to alanine residues leads to complete loss of activity (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005), whereas their replacement with acidic residues induces ectopic anterior expression of Hh target genes, including Engrailed (En) and the ptc-lacZ reporter in wing discs (Jia et al. 2004)(Figure 8C). Also, excess PKA activity induces robust ectopic expression of Hh target genes (wg and ptc) in embryos by a mechanism that requires the activity of both Ci and Smo (Ohlmeyer and Kalderon 1997). On the basis of these results, it might be expected that excess PKA activity can generally activate Smo by direct phosphorylation and hence induce Hh target genes.

Figure 8.—

SmoD1-3 stabilizes Ci-155, promotes Fu phosphorylation, and requires Fu kinase activity to activate Ci strongly. (A, C–E) Expression of full-length Ci-155 detected by antibody 2A1 (red, first column), ptc-lacZ (green, second column), and En protein (green, third column) in wild-type discs (A) and in discs expressing SmoD1-3 alone (C), SmoD1-3 together with activated PKA mC* (D), and SmoD1-3 in the absence of Fu kinase activity (E), using C765-GAL4 at 25°. Ci-155 is selectively stabilized at the AP border of wild-type wing discs (A) but is additionally stabilized in all anterior cells by SmoD1-3 (C). Ci-155 levels induced by SmoD1-3 are greatly reduced by excess PKA without reducing ectopic anterior ptc-lacZ or En induction significantly (D). ptc-lacZ and En induction by SmoD1-3 are drastically reduced in discs hemizygous for the fu^mH63 allele, which encodes kinase-deficient Fu (E). (B) Western blot using antibody to Fu of extracts of wing discs expressing Ci-H5m-w1 (as a control), SmoD1-3, activated PKA (mC*), or mC* together with CK1 (Dbt) using C765-GAL4 at 25°. Only SmoD1-3 strongly induces a change of mobility of Fu characteristic of the hyperphosphorylated species (Fu-p).

However, in wing discs excess PKA has not been shown to induce Hh target genes in the absence of Hh. Rather, only a small enhancement of Hh target gene expression was seen at the AP border (where Hh secreted from posterior cells signals to anterior cells) and this was observed only if the levels of ectopically expressed constitutively active PKA catalytic subunit were low (Jia et al. 2004). Indeed, higher levels of the same activated PKA catalytic subunit reduced the intensity of anterior En induction at the AP border while the intensity of ptc-lacZ staining appeared unchanged (Figure 1, A and B). Thus, maximal Hh pathway activity, which is required for anterior En induction, is reduced by strongly elevated PKA activity. The resulting weaker anterior En expression domain and the ptc-lacZ stripe are broader than normal in these discs, indicating that excess PKA may also enhance the response to low levels of Hh. However, there is clearly no ectopic expression of these Hh target genes in anterior cells far from the AP border in response to excess PKA, even if the CK1 gene doubletime is also overexpressed (Figure 1C).

Figure 1.—

Excess PKA activity inhibits induction of the Hh target gene En in wing discs. Third instar imaginal discs from animals carrying a ptc-lacZ transgene and C765-GAL4 driver only (A) or together with UAS-mC* (B), UAS-mC* and UAS-dbt (C), UAS-Ci-H5m-w1 (D), or both UAS-mC* and UAS-Ci-H5m-w1 (E). Anterior is left; dorsal is up. ptc-lacZ (red) expression in a stripe at the AP border (A) is broadened by excess PKA activity (B and C) and expands into posterior cells when Ci is expressed ubiquitously (D and E). En (green) is expressed in posterior cells independently of Hh but its Hh-dependent expression in anterior cells at the AP border is diminished by excess PKA activity (B) even in the presence of coexpressed Dbt CK1 (C) or Ci that is resistant to silencing by PKA (E). The posterior edge of the AP border ptc-lacZ stripe (indicated by white lines) defines the division between anterior and posterior cells. This compartment border can only be estimated (broken white lines) in D because it is obscured by high levels of ectopic posterior ptc-lacZ expression.

We were concerned that activation of Smo by PKA in the above experiments might have been obscured by coincident inactivation of Ci-155 by direct PKA phosphorylation. We therefore supplemented wing discs with a Ci variant lacking five PKA sites and therefore immune to the direct silencing effects of PKA. The ci transgene, UAS-Ci-H5m-w1 (Price and Kalderon 1999), was expressed ubiquitously at low levels in wing discs using the C765-GAL4 driver so that Ci-H5m-w1 alone did not induce ectopic expression of either of the Hh target genes, ptc-lacZ or En, in anterior cells (Figure 1D). In posterior cells ptc-lacZ was strongly induced by Ci-H5m-w1 under the influence of Hh (Price and Kalderon 1999). Even in the presence of Ci lacking all known functional PKA sites, excess PKA was unable to induce ectopic ptc-lacZ or En expression in anterior wing disc cells away from the AP border (Figure 1E). In fact, excess PKA activity reduced ptc-lacZ expression in posterior cells and also reduced En expression in AP border cells (Figure 1, C and D), indicating reduced pathway activity in response to high levels of Hh.

Full activation of the Hh signaling pathway blocks Ci-155 processing to Ci-75 and also increases the specific activity of Ci-155 (Ohlmeyer and Kalderon 1998; Hooper and Scott 2005). Maximal activation of Ci-155 requires the protein kinase activity of Fused (Fu) and is opposed by Suppressor of fused [Su(fu)]. We therefore tested whether loss of Su(fu) might reveal a subtle or latent ectopic activation of Hh target genes induced by PKA hyperactivity, as was observed previously for slimb and sgg mutant clones (Wang et al. 1999; Jia et al. 2002). However, excess PKA activity also failed to induce ectopic anterior ptc-lacZ or En in the absence of Su(fu) (supplemental Figure S1, A and B, at http://www.genetics.org/supplemental/), even when the Ci-H5m-w1 transgene was also coexpressed (supplemental Figure S1, C and D, at http://www.genetics.org/supplemental/). Instead, just as observed in the presence of Su(fu), excess PKA reduced En expression at the AP border, broadened the ptc-lacZ stripe at the AP border, and reduced ptc-lacZ induction by Ci-H5m-w1 in posterior cells (supplemental Figure S1 at http://www.genetics.org/supplemental/). Thus, despite extensive efforts to expose a stronger effect, excess PKA activity appears to have only a very limited potential to increase Hh target gene expression in wing imaginal discs.

Ci binding sites are required for induction of genes by excess PKA in embryos:

Given the failure of excess PKA activity to activate Hh target genes robustly in wing discs it is surprising that it can activate wg and ptc expression in embryos if this is achieved by phosphorylation and activation of Smo. Indeed, there is already some evidence that the conventional Hh pathway is not strongly activated by excess PKA in embryos because Fu phosphorylation, another measure of Smo activity, is only marginally stimulated (Apionishev et al. 2005). We therefore investigated whether induction of wg and ptc by excess PKA activity in embryos was mediated by Ci binding sites. There is extensive evidence that Ci binding sites in synthetic reporter genes can confer either repression by Ci-75 or activation by Ci-155 and that Ci binding sites normally mediate repression and activation of the key Hh target genes, decapentaplegic (dpp) and ptc, in wing discs (Hepker et al. 1999; Muller and Basler 2000; Methot and Basler 2001). Thus, if PKA hyperactivity induces wg and ptc expression in embryos by activating Smo we would expect Ci binding sites to be the critical feature of the Hh target genes that allows their activation.

Expression of wg in 14 single cell-wide stripes in stage 9–12 embryos is maintained by strong Hh signaling and is limited to the immediate anterior neighbors of Hh-producing cells (Ingham and McMahon 2001). This wg expression pattern can be mimicked by a 5.1-kb regulatory region linked to a lacZ gene (Lessing and Nusse 1998). This reporter, just like the wg gene itself, was expressed beyond the normal range of Hh when either Hh or constitutively active mouse PKA catalytic subunit (mC*) was expressed ectopically in alternating segments using the prd-GAL4 driver (Figure 2, B, G, and L). At the distal end of this 5.1-kb regulatory region is a 1.1-kb fragment that contains several Ci binding sites and that suffices to direct a roughly normal, albeit weak, pattern of expression characteristic of wg (Von Ohlen and Hooper 1997; Von Ohlen et al. 1997). This reporter, Δwg-lacZ, was also expressed in wider stripes in those (alternating) segments of embryos that expressed ectopic Hh, activated PKA (mC*), or a highly expressed transgene (Ci-T5m-s1) encoding Ci lacking key PKA sites (Price and Kalderon 1999) (Figure 2, D, I, and N). The equivalent reporter gene (Δwg*-lacZ), in which the Ci binding sites have been altered by point mutations to eliminate Ci binding in vitro, is barely expressed at all in wild-type embryos, demonstrating the critical role of Ci binding sites in responding to Hh (Von Ohlen and Hooper 1997). A few embryos expressed Δwg*-lacZ in response to expression of the highly active Ci-T5m-s1 transgene in alternating segments (Figure 2O). Most embryos expressing mC* in alternating segments showed no Δwg*-lacZ expression, although very weak expression in thin stripes was detected in a few embryos (Figure 2J). Thus, a 1.1-kb segment of the wg enhancer is sufficient to respond to PKA hyperactivity and this response is largely contingent on the presence of functional Ci binding sites. The trace induction of Δwg*-lacZ by excess PKA likely corresponds to residual binding of Ci to this reporter since expression of an activated form of Ci also induced Δwg*-lacZ weakly.

Figure 2.—

Ci binding sites are required for induction of wg reporter genes by PKA hyperactivity in embryos. In situ RNA hybridization of wg (A, F, and K) and lacZ (B–E, G–J, and L–O) probes to stage 11–12 embryos that are wild type (A–E) or that express activated PKA catalytic subunit mC* (F–J), ectopic Hh (K–M), or ectopic activated Ci, Ci-T5m-s1 (N and O) in alternating segments using prd-GAL4. Representative segments on the ventral surface (down) that express the transgenes are indicated by filled boxes aligned underneath. Expression of lacZ is from the reporter genes wg5.1-lacZ (B, G, and L), WLZ Gc2.5L (C, H, and M), Δwg-lacZ (D, I, and N), or Δwg*-lacZ (E, J, and O). Stripes of expression of wg, wg5.1-lacZ, and Δwg-lacZ (which all contain Ci-binding sites) are expanded and enhanced by mC* (F, G, and I) and Hh or activated Ci (K, L, and N). Expression of WLZ Gc2.5L and Δwg*-lacZ (both of which lack identified strong Ci-binding sites) is not affected by mC* (H and J). WLZ Gc2.5L expression is induced by Hh (M), whereas Δwg*-lacZ is barely expressed in wild-type embryos (E) and is induced only in a few embryos by activated Ci (O). Anterior is to the left and dorsal is up.

There is a precedent for induction of wg by Hh through enhancer elements other than Ci binding sites. A regulatory element within the wg 5.1-kb enhancer, named “box G,” contains no discernible Ci binding sites but was previously shown to repress expression of a reporter gene and to confer derepression in response to Hh signaling (Lessing and Nusse 1998). A reporter (WLZGc2.5L) containing two copies of box G but lacking the 1.1-kb Ci binding site region of the 5.1-kb enhancer was indeed induced ectopically by Hh, but it was not induced by excess PKA (Figure 2, C, H, and M). Thus, analysis of wg regulatory regions suggests that Ci binding sites are key elements required to respond to PKA hyperactivity.

Ci binding sites suffice for only a small response to excess PKA in embryos:

We then tested whether Ci binding sites suffice for induction of a reporter gene by PKA hyperactivity in stage 9–12 embryos. Barolo and Posakony (2002) established a general principle that signaling pathways induce artificial reporter genes efficiently if binding sites for the transcriptional effector of the pathway are combined with binding sites for a transcriptional activator that is ubiquitous and constitutively active. Thus, a reporter with four Ci binding sites (4xCi-lacZ) is barely expressed in wing discs (Barolo and Posakony 2002) or in wild-type embryos; it is not responsive to ectopic Hh and can be weakly induced only by expression of excess Ci from the Ci-T5m-s1 transgene (data not shown). However, a reporter containing four Ci binding sites adjacent to three binding sites for the transcriptional activator Grainyhead (Ci-Grh-lacZ) is expressed much like the ptc gene in wing discs (Barolo and Posakony 2002) and in stage 9–12 embryos, in roughly single cell-wide stripes either side of each stripe of Hh expression (Figure 3, A and D). Ectopic Hh expression in all anterior cells, achieved using ptc-GAL4 together with UAS-Hh, expanded Ci-Grh-lacZ reporter expression to most anterior cells (Figure 3C) [strong ectopic induction of wg by ectopic Hh leads to ectopic En expression in adjacent cells, preventing induction of Hh target genes in those En-expressing cells (Bejsovec and Wieschaus 1993)]. However, only a very small expansion in the domain of Ci-Grh-lacZ expression was elicited by expression of activated PKA (mC*) using ptc-GAL4 (Figure 3, B and E). This expansion was much less than that observed for wg mRNA in equivalent embryos processed in parallel (Figure 3, H and I). Thus, Ci binding sites taken out of their normal context in the wg enhancer respond strongly to Hh but only very weakly to excess PKA activity.

Figure 3.—

Minimal induction of a reporter gene by PKA hyperactivity through Ci binding sites alone. In situ RNA hybridization to stage 11–12 embryos carrying a Ci-Grh-lacZ reporter gene using a lacZ probe (A–G) or a wg probe (H–K). (A–C) Lateral views (anterior left, dorsal up) showing repeated pairs of stripes of reporter gene expression (blue) corresponding to cells either side of thin intervening stripes of Hh-expressing cells in wild-type embryos (A). Ci-Grh-lacZ expression is largely unchanged in response to mC* expression driven by ptc-GAL4 (B) but stripes of expression are expanded in response to ectopic Hh driven by ptc-GAL4 (C). (D–K) Ventral surface showing slight, patchy broadening of Ci-Grh-lacZ expression by mC* driven by ptc-GAL4 (E) relative to wild type (D). This broadening is considerably enhanced by loss of zygotic Su(fu) (G), which has no effect alone (F). mC* expression induces a strong expansion of wg stripes in the presence (I vs. H) and absence (K vs. J) of zygotic Su(fu) activity.

We considered the possibility that Ci-Grh-lacZ might be less sensitive than wg to Hh pathway activity and that excess PKA might therefore activate the Hh pathway to a level just below the threshold required to induce Ci-Grh-lacZ, leading to a deceptively small response. We tested this possibility in two ways. First, we compared the response of Ci-Grh-lacZ to excess PKA and to PKA inhibition. PKA inhibition induces wg mRNA less strongly than does excess PKA activity or excess Hh (Ohlmeyer and Kalderon 1997) (Figure 4, D–F), consistent with weak activation of the Hh pathway effector Ci. Nevertheless, Ci-Grh-lacZ was clearly induced by PKA inhibition, contrasting with the negligible induction by excess PKA activity (Figure 4, A–C). Thus, Ci-Grh-lacZ is clearly proportionally less responsive than wg to PKA hyperactivity when compared to either weak activation of the Hh pathway by PKA inhibition or strong activation by Hh itself.

Figure 4.—

Ci lacking PKA sites does not enhance the response of a reporter gene to excess PKA activity through Ci binding sites. Expression of Ci-Grh-lacZ (shown in lateral view, A–C and G–I, anterior left) and wg (shown in ventral view, D–F and J–L) in wild-type embryos (A and D) and in embryos expressing Ci lacking five PKA sites, Ci-H5m-w1 (G and J), activated PKA catalytic subunit mC* (B and E), PKA inhibitor R* (C and F), both mC* and Ci-H5m-w1 (H and K), or both R* and Ci-H5m-w1 (I and L) in alternating segments using prd-GAL4. Representative segments expressing (filled boxes) and not expressing (open boxes) the transgenes are indicated. PKA inhibition with R* in alternating segments produces a patchy expansion of the thinner anterior Ci-Grh-lacZ stripe in an anterior direction toward the stronger posterior Ci-Grh-lacZ stripe of the next segment (C). mC* causes a similar but weaker ectopic induction of Ci-Grh-lacZ, most obvious as an alternating reduction in the unstained space between adjacent sets of paired stripes (B). Ci-H5m-w1 expression induces ectopic Ci-Grh-lacZ expression in the Hh-producing cells between each pair of stripes in alternating segments (G) and enhances the anterior expansion of Ci-Grh-lacZ expression due to R* (I) but does not produce significant anterior expansions alone (G) or enhance those due to mC* expression (H). mC* induces a greater expansion of wg stripes than does R*, both in the absence (D–F) and presence of coexpressed Ci-H5m-w1 (J–L).

Second, we tried to alter the threshold of the response of Ci-Grh-lacZ by augmenting Hh pathway activity to see if this revealed a stronger response to excess PKA activity. We first tried to do this by coexpressing low levels of Ci lacking PKA sites (Ci-H5m-w1), which by itself produces no ectopic activation of wg or Ci-Grh-lacZ (Figure 4, G and J). However, we still observed only a very small expansion of Ci-Grh-lacZ expression in alternating segments of embryos that expressed both Ci-H5m-w1 and mC* (Figure 4H). By contrast, Ci lacking PKA sites clearly increased the response of Ci-Grh-lacZ to PKA inhibition (Figure 4I). We then tested the consequences of inactivating Su(fu). Loss of Su(fu) by itself does not alter the expression of either wg or Ci-Grh-lacZ in embryos but it did increase the response of Ci-Grh-lacZ to PKA hyperactivity (Figure 3, F and G). Thus, under some conditions PKA hyperactivity can induce clear ectopic activation of a synthetic Hh pathway reporter containing repeated consensus Ci binding sites. However, even in the absence of Su(fu) excess PKA induced the Ci-Grh-lacZ reporter less strongly than did inhibition of PKA (not shown) and the Ci-Grh-lacZ reporter was induced much less strongly than the wg gene itself (Figure 3, J and K). Thus, when tested under a variety of conditions and compared to Hh itself or activation of Ci via PKA inhibition, excess PKA induces a reporter containing only Ci binding sites much less well than it induces wg or ptc. This suggests that excess PKA does not induce wg expression in embryos solely through activation of Ci.

Sequences distant from critical Ci binding sites in the ptc gene mediate induction by excess PKA:

The ability of wg and ptc genes to respond to PKA hyperactivity in embryos more strongly than a Ci binding site reporter might be due to a specific local arrangement or context of Ci binding sites or, alternatively, to distinct additional sequences that collaborate with Ci binding sites to increase induction by excess PKA. For the wg gene the strong response of the Δwg-lacZ reporter (Figure 2) indicates that sufficient critical sequences lie on a 1.1-kb fragment. For ptc, a 12-kb regulatory region driving lacZ (ptc-lacZ) faithfully reflects the normal regulation of the ptc gene in embryos and imaginal discs and the most proximal 800 bp of this region, which includes three identified Ci binding sites, suffices to direct a stripe of FE-lacZ reporter expression at the AP border of wing discs, as seen for the endogenous ptc transcript (Forbes et al. 1993; Alexandre et al. 1996). In embryos, ptc-lacZ and FE-lacZ are expressed either side of Hh-expressing stripes and their expression expanded to include most anterior cells when Hh was ectopically expressed using ptc-GAL4 (Figure 5). PKA hyperactivity strongly induced ptc-lacZ expression throughout anterior regions of each segment but did not detectably affect the expression pattern of FE-lacZ (Figure 5, B and E). The FE-lacZ reporter is expressed more weakly than ptc-lacZ or Ci-Grh-lacZ reporters in response to Hh, so subtle induction of this reporter by excess PKA could be missed. Nevertheless, we can conclude that the clustered Ci sites within ptc regulatory sequences do not suffice to respond strongly to excess PKA activity in embryos, implying that regulatory sequences in the 11 kb upstream of the known Ci binding sites in the ptc gene are instrumental in responding to PKA.

Figure 5.—

Induction of ptc reporters by PKA hyperactivity requires regulatory sequences beyond the proximal Ci binding sites. Lateral views (anterior to left) of embryos carrying the FE-lacZ (A–C and G–I) or ptc-lacZ reporter genes (D–I), showing expression of lacZ (A–F) and ptc RNAs (G–I). FE-lacZ, ptc-lacZ, and ptc RNA are all expressed in the same pattern of a pair of stripes straddling each stripe of Hh expression in wild-type embryos (A, D, and G) and all stripes are expanded to include most anterior cells when Hh is ectopically expressed using ptc-GAL4 (C, F, and I). However, expression of mC* using ptc-GAL4 greatly expands the expression domain of ptc and ptc-lacZ RNAs (E and H), but leaves FE-lacZ expression unaffected (B).

Is induction of wg and ptc by PKA hyperactivity in embryos mediated by Smo phosphorylation?

The analysis of regulatory elements described above shows that the response of embryos to excess PKA activity is captured poorly by Ci binding sites alone and is therefore unlikely to be mediated solely by activation of Smo. We wished to test the role of Smo phosphorylation in responding to PKA hyperactivity more directly by altering the PKA sites in Smo that are known to affect Smo activity. We initially tried to do this by substituting wild-type Smo with a Smo variant where the three PKA sites in question were altered to alanine residues. However, this variant lost all activity in response to both PKA and Hh; hence we could not distinguish whether PKA acts through Smo or separately but with a required additional input from Smo activity (in parallel) (Apionishev et al. 2005).

To circumvent this ambiguity we tested whether a Smo variant in which the PKA sites and consensus PKA-primed and CK1-primed CK1 sites were changed to aspartate residues (SmoD1-3) (Jia et al. 2004) could respond to PKA and Hh in embryos. Expression of SmoD1-3 (using prd-GAL4) in alternating segments of embryos lacking endogenous maternal and zygotic smo activity rescued single cell-wide stripes of wg and ptc in the segments where SmoD1-3 was expressed, but did not induce ectopic expression of wg or ptc beyond the normal Hh signaling domain (Figure 6, B and F). Thus, at the levels of expression employed, SmoD1-3 does not have detectable constitutive activity in embryos but transduces an Hh signal efficiently. This was confirmed by coexpressing Hh (using prd-GAL4), resulting in an expansion of wg and ptc RNA stripes to roughly the anterior limit of prd-GAL4 expression in alternating segments (Figure 6, D and H). Coexpression of SmoD1-3 with mC* to increase PKA activity in embryos lacking wild-type Smo produced a similar strong expansion of wg and ptc RNA stripes in the prd-GAL4 expression pattern (Figure 6, C and G). Thus, PKA hyperactivity can induce wg and ptc strongly in the absence of critical PKA target sites in Smo. We therefore conclude that the major mechanism for the induction of Hh target genes by excess PKA in embryos is not driven by PKA phosphorylation of Smo.

Figure 6.—

PKA hyperactivity strongly induces wg and ptc expression without phosphorylating Smo. Lateral views (anterior to left) of wild-type embryos (A and E), and embryos lacking maternal and zygotic smo activity (due to the smo² allele) but expressing SmoD1-3 (B and F), SmoD1-3 and mC* (C and G), or SmoD1-3 and Hh (D and H) in alternating segments using prd-GAL4. Expression of wg is rescued by SmoD1-3 in alternating stripes of normal width (B). These rescued stripes are greatly expanded by coexpression of mC* (C) or Hh (D). Similarly, pairs of ptc stripes are rescued in alternating segments by SmoD1-3 (F) and expanded by coexpression of mC* (G) or Hh (H).

Role of Smoothened phosphorylation by PKA in wing discs:

Given that excess PKA activity cannot readily activate the Hh pathway via Smo in embryos or wing discs, it is important to reconsider whether normal PKA activity is required for Smo to respond to Hh. Substitution of alanine residues at PKA and CK1 sites on Smo produces complete inactivation in wing discs (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). However, loss of activity of the major PKA catalytic subunit PKA-C1 still allows strong Smo-dependent induction of the Hh-target gene collier at the AP border of wing discs, provoking the suggestion that enzymes other than PKA-C1 might contribute to phosphorylation of Smo at PKA sites (Apionishev et al. 2005). We therefore investigated whether there was any requirement for Smo phosphorylation by PKA in wing discs.

There is prior evidence from assaying wing margin bristle phenotypes, and from measuring induction of a synthetic 4bs-lacZ reporter gene and anterior En expression at the AP border, that PKA-C1 does contribute positively to the outcome of Hh signaling in wing discs (Jiang and Struhl 1995; Ohlmeyer and Kalderon 1998; Wang and Holmgren 2000; P. Therond, personal communication). We tested if this positive role of PKA was due to phosphorylation of Smo by trying to complement the PKA-C1 mutant defect with SmoD1-3. Anterior PKA-C1 mutant clones induce a low level of ectopic En cell autonomously (Ohlmeyer and Kalderon 1998) but they also clearly reduced the level of En normally induced by Hh at the AP border (Figure 7A). Expression of SmoD1-3 at low levels (using C765-GAL4 at 18°) induced some patchy ectopic anterior En expression but only at levels clearly lower than at the AP border. Under these conditions SmoD1-3 rescued normal high levels of En at the AP border in the absence of endogenous Smo activity (Figure 7B), showing that SmoD1-3 activity can be increased by Hh in wing discs, as in embryos. SmoD1-3 also rescued normal levels of En at the AP border in clones that lacked both endogenous Smo and PKA-C1 activities (Figure 7C). Thus, mimicking Smo phosphorylation with acidic residues eliminates the deficit in Hh signaling caused by loss of PKA-C1 activity. This is consistent with the idea that PKA-C1 must phosphorylate Smo for Hh to signal optimally at the AP border of wing discs.

Figure 7.—

SmoD1-3 restores En expression to PKA mutant clones at the AP border. (A–C) Third instar wing discs (anterior left, dorsal up) stained for En protein (red), GFP (green) to mark homozygous mutant clones lacking GFP, and ptc-lacZ expression using antibodies to β-galactosidase (blue). En expression at the AP border is substantially lost in PKA-C1 mutant clones (arrowhead, A) but is restored to both smo mutant clones (arrowhead, B) and smo PKA-C1 double mutant clones (arrowhead, C) at the AP border by expression of UAS-SmoD1-3 driven by C765-GAL4 at 18°. In each case anterior En expression is distinguished from posterior En expression by its overlap with ptc-lacZ expression, which is strictly confined to anterior cells. White lines mark the posterior limit of ptc-lacZ expression. Note that the levels of En at the AP border are higher than induced in far anterior cells by either SmoD1-3 or PKA-C1 mutant clones alone but similar to levels in far anterior PKA-C1 clones that also express SmoD1-3.

Does Smo phosphorylation at PKA and CK1 sites affect all aspects of Hh signaling?

Hh signaling blocks processing of Ci-155 to Ci-75 and also increases transcriptional activation by Ci-155 (Hooper and Scott 2005). The activity of Ci-155 can be affected by several pathway components, such as PKA and Cos2, that also affect Ci-155 processing. However, loss of Fu kinase diminishes Ci-155 activity in response to Hh and loss of Su(fu) can potentiate Ci-155 activity without significantly affecting Ci-155 processing (Alves et al. 1998; Ohlmeyer and Kalderon 1998). Hence, it is often suggested that Smo can initiate at least two distinct biochemical pathways, one affecting Ci-155 processing and another affecting Ci-155 specific activity via Fused. Since SmoD1-3 only partially activates the Hh pathway and can be further activated by Hh it was important to test whether mimicking Smo phosphorylation activates each of the postulated biochemical pathways downstream of Smo.

We found that ubiquitous expression of SmoD1-3 greatly increased Ci-155 levels in anterior cells of wing discs, so that the normal accentuation of Ci-155 staining at the AP border was no longer discernible (Figure 8, A and C). This is consistent with inhibition of Ci-155 processing to Ci-75. SmoD1-3 expression also increased Fu phosphorylation substantially, as measured in extracts of wing discs (Figure 8B), as observed previously for a similar Smo variant in cultured cells (Zhang et al. 2004). Hyperphosphorylation of Fu also occurs in cells responding to Hh and may reflect activation of Fu (Hooper and Scott 2005). More importantly, in wing discs that lack Fu kinase activity the strong induction of ptc-lacZ in anterior cells by SmoD1-3 (using C765-GAL4 at 25°) was greatly curtailed and ectopic induction of En by SmoD1-3 was completely eliminated (Figure 8E). Thus, SmoD1-3 must constitutively activate the branch of the Hh pathway that utilizes the kinase activity of Fu.

Further evidence that SmoD1-3 not only stabilizes Ci-155 but also increases its specific activity comes from experiments in which SmoD1-3 was ubiquitously expressed together with activated PKA (mC*). mC* coexpression greatly reduced levels of full-length Ci-155 in anterior and AP border cells (Figure 8, C and D), as might be expected as a consequence of increased Ci phosphorylation, but did not significantly reduce ectopic anterior expression of either ptc-lacZ or En (Figure 8, C and D). Thus, it appears that two arguably separate aspects of Hh signaling are phenocopied by changing Smo PKA and CK1 sites to acidic residues.

DISCUSSION

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 (Jiang and Struhl 1995; Lepage et al. 1995; Li et al. 1995; Pan and Rubin 1995). 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 (Jia et al. 2005; Pan et al. 2006; Smelkinson and Kalderon 2006; Wang and Li 2006). 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 (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). Those and other studies left open a number of significant questions. 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?

Must Smo be phosphorylated by PKA for Hh to signal?

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 (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). 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 (Apionishev et al. 2005). However, PKA inhibition with R* in embryos does not prevent all Hh-stimulated phosphorylation of Fu or Hh-dependent maintenance of wg expression (Ohlmeyer and Kalderon 1997; Apionishev et al. 2005). 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 (Lane and Kalderon 1993; Lane and Kalderon 1994; Ohlmeyer and Kalderon 1997; Apionishev et al. 2005).

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 (Lane and Kalderon 1993; Melendez et al. 1995). 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 (Jia et al. 2004; P. Therond, personal communication; this study). We found that 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 (Apionishev et al. 2005). 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 (Apionishev et al. 2005) but not when PKA-C1 activity is eliminated (Nakano et al. 2004; Apionishev et al. 2005).

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 (Methot and Basler 2000; Hooper and Scott 2005). 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 (Zhang et al. 2005). However, Cos2 also binds to Smo (Hooper and Scott 2005) 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 (Jia et al. 2005; Smelkinson and Kalderon 2006). 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 we speculate that a low rate of phosphorylation of Smo at PKA sites may suffice for it to be active.

Is phosphorylation of Smo at PKA and CK1 sites sufficient to activate the Hh pathway?

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 (Jia et al. 2004; Zhang et al. 2004; Hooper and Scott 2005). Our 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.

First, we 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 and was described previously (Jia et al. 2004). 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. We speculate 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.

Second, we found that excess PKA activity and CK1 activity cannot reproduce the ectopic activation of Hh target genes induced by expression of SmoD1-3 (Figure 1 and supplemental Figure S1 at http://www.genetics.org/supplemental/). This was true despite our 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 (Figure 8).

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. We do not favor this explanation because both of the protein kinases used are thought to associate with Cos2 (Zhang et al. 2005) 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 (Price and Kalderon 2002).

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 we excluded this confounding influence 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 we 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.

Finally, our favored explanation is that Smo with acidic residues at PKA and CK1 sites behaves significantly differently from Smo that is phosphorylated at those sites. We have previously 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 (Apionishev et al. 2005). We further speculate 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.

A significant question for the future is how phosphorylation of Smo contributes to its activity. We have some clues 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 (Ogden et al. 2004; Hooper and Scott 2005). 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.

Induction of Hh target genes in embryos by factors other than Ci:

We 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 (i) detectable, but very limited, ectopic expression of a reporter gene that essentially contains only Ci binding sites (Figures 3 and 4), (ii) clear ectopic expression of a wg reporter gene that depends on the presence of Ci binding sites (Figure 1), and (iii) a small increase in Fu phosphorylation (Apionishev et al. 2005). 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 (Figures 2–5) 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 (Figure 6).

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.

PKA is known to phosphorylate many proteins that can influence transcription (Conkright et al. 2003; Rochette-Egly 2003; Martin et al. 2004; Poels and Vanden Broeck 2004) 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 (Lessing and Nusse 1998; Gallet et al. 2000; Muller and Basler 2000). 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 (Muller and Basler 2000; Hooper and Scott 2005). Whether Hh signaling affects the activity or interactions of transcription factors that collaborate with Ci is not presently known.

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 (Ohlmeyer and Kalderon 1997) and requires functional Ci binding sites within the Δwg-lacZ reporter gene (Figure 2). Even the PKA sites on Smo are required for wg to respond to excess PKA (Apionishev et al. 2005), consistent with the idea that some activation of Smo is required. We do not yet, however, have any 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 (Barolo and Posakony 2002) 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 (Hooper 1994; Yoffe et al. 1995). 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 (data not shown) and PKA hyperactivity also induces ectopic ptc expression, which does not depend on Wg activity for its expression (data not shown). 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.