the contributions of protein kinase a and smoothened

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.061036

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

Qianhe Zhou,1 Sergey Apionishev1 and Daniel Kalderon2

Department of Biological Sciences, Columbia University, New York, New York 10027

Manuscript received May 19, 2006Accepted for publication June 7, 2006

ABSTRACT

Protein kinase A (PKA) silences the Hedgehog (Hh) pathway in Drosophila in the absence of ligand byphosphorylating the pathway’s transcriptional effector, Cubitus interruptus (Ci). Smoothened (Smo) isessential for Hh signal transduction but loses activity if three specific PKA sites or adjacent PKA-primedcasein kinase 1 (CK1) sites are replaced by alanine residues. Conversely, Smo becomes constitutively activeif acidic residues replace those phosphorylation sites. These observations suggest an essential positive rolefor PKA in responding to Hh. However, direct manipulation of PKA activity has not provided strongevidence for positive effects of PKA, with the notable exception of a robust induction of Hh target genesby PKA hyperactivity in embryos. Here we show that the latter response is mediated principally byregulatory elements other than Ci binding sites and not by altered Smo phosphorylation. Also, the failureof PKA hyperactivity to induce Hh target genes strongly through Smo phosphorylation cannot beattributed to the coincident phosphorylation of PKA sites on Ci. Finally, we show that Smo containingacidic residues at PKA and CK1 sites can be stimulated further by Hh and acts through Hh pathways thatboth stabilize Ci-155 and use Fused kinase activity to increase the specific activity of Ci-155.

THE Hedgehog (Hh) signaling pathway regulatesmany aspects of cell specification and cell pro-

liferation in organisms from Drosophila to humans andis of prime medical importance, especially because itsgenetic 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 aresome indications of significant differences in the mech-anism of Hh signal transduction between Drosophilaand vertebrate cells (Huangfu and Anderson 2006;Svard et al. 2006; Varjosalo et al. 2006), there is alsoclearly a great deal of conservation, and the bulk of ourcurrent understanding derives from studies in Dro-sophila (Hooper and Scott 2005). Key conservedcomponents include the Hh receptor Patched (Ptc),the transmembrane protein Smoothened (Smo), andthe transcriptional effector Cubitus interruptus (Ci;homologous to Gli proteins in vertebrates). In all casesPtc silences the pathway in the absence of ligand byallowing processing of Ci or Gli proteins to forms thatact as transcriptional repressors, while limiting the ac-cumulation and activity of full-length Ci or Gli tran-scriptional activators. When Hh family ligands bind toPtc the processing of Ci/Gli proteins is blocked and

these proteins instead become effective transcriptionalactivators. Smo is required to mediate these responsesto Hh ligands but neither the regulation of Smo activityby Ptc and Hh nor the effector functions of Smo arewell understood (Hooper and Scott 2005).

Protein phosphorylation is instrumental in both thesilencing and the activation of the Hh pathway. Pro-teolytic 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-primedcasein kinase 1 (CK1) and glycogen synthase kinase 3(GSK3) sites (Aza-Blanc et al. 1997; Jia et al. 2002; Priceand Kalderon 2002). Phosphorylation at these andfurther primed sites by these three protein kinases cre-ates a binding site for the Skp1/Cullin1/F-box compo-nent Slimb (Jia et al. 2005; Smelkinson and Kalderon2006). This presumably leads to Ci-155 ubiquitinationand partial proteolysis by the proteasome (Maniatis

1999; Tian et al. 2005). Vertebrate Gli proteins includeanalogous PKA, CK1, and GSK3 sites and the proteolysisof Gli3 and Gli2 depends on these sites and the con-sequent binding of the Slimb homolog b-TRCP (Panet al. 2006; Wang and Li 2006). Furthermore, Hh sig-naling can regulate the partial proteolysis of Gli3 pro-tein, both in vertebrates and when Gli3 is introducedinto Drosophila (von Mering and Basler 1999; Aza-Blanc et al. 2000; Wang et al. 2000). In addition to pro-moting repressor formation, phosphorylation may alsolimit the specific activity of Ci-155 as a transcriptional

1These authors contributed equally to this work.2Corresponding author: Department of Biological Sciences, 1013 Fair-

child, MC 2445, Columbia University, 1212 Amsterdam Ave., New York,NY 10027. E-mail: [email protected]

Genetics 173: 2049–2062 (August 2006)

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activator (Wang et al. 1999). Through these actions onCi/Gli proteins PKA silences the Hh pathway in Dro-sophila and vertebrates in the absence of ligand.

Phosphorylation also affects Smo activity. DrosophilaSmo requires a cluster of three PKA sites and the ad-jacent 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 reducedby inhibiting PKA and CK1 activities but this deficit insignaling is hard to measure and evaluate accuratelybecause loss of PKA or CK1 simultaneously contributesto activation of Ci-155 independent of any input fromHh or Smo (Jia et al. 2004; Apionishev et al. 2005).Alteration of all of the clustered PKA and adjacentprimed CK1 sites in Smo to acidic residues, potentiallymimicking phosphorylation, confers some Hh-indepen-dent activity on Smo (Jia et al. 2004; Zhang et al. 2004).Hence, Smo phosphorylation on PKA and CK1 sites isnecessary for activity and may even suffice to activateSmo. These critical PKA and CK1 sites are not conservedin vertebrate Smo proteins, which have smaller carboxy-terminal cytoplasmic domains, implying a notable dif-ference in the design of vertebrate and invertebrate Hhpathways (Huangfu and Anderson 2006; Varjosaloet al. 2006). There is, however, some evidence thatHh-dependent phosphorylation of vertebrate Smo atG-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 forphosphorylation of Smo at defined PKA and CK1 sites inHh pathway activation there are a number of apparentinconsistencies in the data supporting this idea, in-cluding the markedly different potencies of excess PKAactivity in inducing Hh target genes in embryos com-pared to wing discs (Ohlmeyer and Kalderon 1997;Jia et al. 2004). Here we resolve some of these incon-sistencies. In doing so, we find that strong induction ofHh target genes in embryos by excess PKA activitydepends on regulatory elements other than Ci bindingsites and we explore further how Smo phosphorylationaffects Hh pathway activity.

MATERIALS AND METHODS

Immunohistochemistry and RNA in situ hybridization:Embryo in situ hybridization was performed as describedpreviously (Ohlmeyer and Kalderon 1997) using digoxige-nin-labeled RNA probes for wg, ptc, and E. coli lacZ geneproducts described previously (Ohlmeyer and Kalderon1997; Lessing and Nusse 1998). Third instar wing discs werestained as in Smelkinson and Kalderon (2006) with rabbitanti-b-galactosidase and mouse anti-En monoclonal 4D9(DSHB), using AlexaFluor594 and AlexaFluor488 seconda-ries, respectively, or AlexaFluor647 for En staining when GFPwas 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 Hhon the wg gene reporters wg-lacZ5.1, WLZGc2.5L (Lessing andNusse 1998), Dwg-lacZ, Dwg*-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 reporterCi-Grh-lacZ (Barolo and Posakony 2002) were assayed in thefollowing crosses (where RG1 and ptc-GAL4 are enhancer trapinsertions of a GAL4 transgene; Ohlmeyer and Kalderon1997):

1. RG1 (¼ prd-GAL4)/TM3 crossed to UAS-mC*; wg-lacz5.1, toUAS-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, toUAS-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 Dwg-lacZ; RG1/TM3 or Dwg*-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 orto UAS-mC*; Su(fu)LP.

Wg requirements for wg and ptc reporter expression inembryos were assayed in wgcx4 ptcS2/CyO ftz-lacZ; wg-lacZ5.1 andwgcx4 ptcS2/CyO ftz-lacZ; FE-lacZ/TM2 stocks and in crosses ofwgcx4 UAS-mC*/CyO; wg-lacZ5.1 to wgcx4/CyO; RG1/TM2 stocks.

SmoD1-3 was expressed in alternating segments of smomutant embryos, together with Hh or mC* in crosses of yhs-flp/yw; smo2 ck FRT40A/ P[ovo]D FRT40A; RG1/1 females thatwere heat-shocked for 1 hr at 37� as third instar larvae to malesthat 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 forsmo2 (and lacking germline smo activity) were readily identifiedas lacking wg and ptc expression in segments that do notexpress 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 ofsmo and PKA-C1 mutant clone phenotypes by SmoD1-3 wastested in animals of the following genotypes heat-shocked atsecond 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-C1B3 FRT40A/Ubi-GFPFRT40A; C765-GAL4 ptc-lacZ/UAS-SmoD1-3, and yw hs-flp/yw;ck PKA-C1B3 FRT40A/Ubi-GFP FRT40A; C765-GAL4 ptc-lacZ/1.Discs expressing SmoD1-3 but lacking Fu kinase activity weredissected from y male larvae from the cross of yw fumH63/ywfumH63; P[y1] P[Fu1]/CyO; C765-GAL4 ptc-lacZ/1 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/1, UAS-mC*/1; C765-GAL4 ptc-lacZ/1, UAS-mC*/1; C765-GAL4 ptc-lacZ/ UAS-dbt, orC765-GAL4 ptc-lacZ/UAS-SmoD1-3 larvae raised at 29�.

RESULTS

Can excess PKA activity induce Hh pathway activityin wing discs? Conversion of PKA and CK1 sites on Smoto alanine residues leads to complete loss of activity (Jiaet al. 2004; Zhang et al. 2004; Apionishev et al. 2005),whereas their replacement with acidic residues inducesectopic anterior expression of Hh target genes, includ-ing Engrailed (En) and the ptc-lacZ reporter in wing

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discs (Jia et al. 2004)(Figure 8C). Also, excess PKAactivity induces robust ectopic expression of Hh targetgenes (wg and ptc) in embryos by a mechanism thatrequires the activity of both Ci and Smo (Ohlmeyer andKalderon 1997). On the basis of these results, it mightbe expected that excess PKA activity can generallyactivate Smo by direct phosphorylation and hence in-duce Hh target genes.

However, in wing discs excess PKA has not beenshown to induce Hh target genes in the absence of Hh.Rather, only a small enhancement of Hh target geneexpression was seen at the AP border (where Hhsecreted from posterior cells signals to anterior cells)and this was observed only if the levels of ectopicallyexpressed constitutively active PKA catalytic subunitwere low (Jia et al. 2004). Indeed, higher levels of thesame activated PKA catalytic subunit reduced the in-

tensity of anterior En induction at the AP border whilethe 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 reducedby strongly elevated PKA activity. The resulting weakeranterior En expression domain and the ptc-lacZ stripeare broader than normal in these discs, indicating thatexcess PKA may also enhance the response to low levelsof Hh. However, there is clearly no ectopic expression ofthese Hh target genes in anterior cells far from the APborder in response to excess PKA, even if the CK1 genedoubletime is also overexpressed (Figure 1C).

We were concerned that activation of Smo by PKA inthe above experiments might have been obscured bycoincident inactivation of Ci-155 by direct PKA phos-phorylation. We therefore supplemented wing discswith a Ci variant lacking five PKA sites and therefore

Figure 1.—Excess PKA activity inhibits induc-tion of the Hh target gene En in wing discs. Thirdinstar imaginal discs from animals carrying a ptc-lacZ transgene and C765-GAL4 driver only (A)or together with UAS-mC* (B), UAS-mC* andUAS-dbt (C), UAS-Ci-H5m-w1 (D), or both UAS-mC* and UAS-Ci-H5m-w1 (E). Anterior is left; dor-sal is up. ptc-lacZ (red) expression in a stripe atthe AP border (A) is broadened by excess PKA ac-tivity (B and C) and expands into posterior cellswhen Ci is expressed ubiquitously (D and E). En(green) is expressed in posterior cells indepen-dently of Hh but its Hh-dependent expressionin anterior cells at the AP border is diminishedby excess PKA activity (B) even in the presenceof coexpressed Dbt CK1 (C) or Ci that is resistantto silencing by PKA (E). The posterior edge ofthe AP border ptc-lacZ stripe (indicated by whitelines) defines the division between anterior andposterior cells. This compartment border canonly be estimated (broken white lines) in D be-cause it is obscured by high levels of ectopic pos-terior ptc-lacZ expression.

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immune to the direct silencing effects of PKA. The citransgene, UAS-Ci-H5m-w1 (Price and Kalderon 1999),was expressed ubiquitously at low levels in wing discsusing the C765-GAL4 driver so that Ci-H5m-w1 alonedid not induce ectopic expression of either of theHh target genes, ptc-lacZ or En, in anterior cells (Figure1D). In posterior cells ptc-lacZ was strongly inducedby Ci-H5m-w1 under the influence of Hh (Price andKalderon 1999). Even in the presence of Ci lacking allknown functional PKA sites, excess PKA was unable toinduce ectopic ptc-lacZ or En expression in anterior wingdisc cells away from the AP border (Figure 1E). In fact,excess PKA activity reduced ptc-lacZ expression inposterior cells and also reduced En expression in APborder cells (Figure 1, C and D), indicating reducedpathway 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 specificactivity 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)]. Wetherefore tested whether loss of Su(fu) might reveal asubtle or latent ectopic activation of Hh target genesinduced by PKA hyperactivity, as was observed previ-ously for slimb and sgg mutant clones (Wang et al. 1999;Jia et al. 2002). However, excess PKA activity alsofailed to induce ectopic anterior ptc-lacZ or En in theabsence of Su(fu) (supplemental Figure S1, A and B, athttp://www.genetics.org/supplemental/), even whenthe Ci-H5m-w1 transgene was also coexpressed (supple-mental Figure S1, C and D, at http://www.genetics.org/supplemental/). Instead, just as observed in the pres-ence of Su(fu), excess PKA reduced En expression atthe AP border, broadened the ptc-lacZ stripe at the APborder, and reduced ptc-lacZ induction by Ci-H5m-w1 inposterior cells (supplemental Figure S1 at http://www.genetics.org/supplemental/). Thus, despite extensiveefforts to expose a stronger effect, excess PKA activityappears to have only a very limited potential to increaseHh target gene expression in wing imaginal discs.

Ci binding sites are required for induction of genesby excess PKA in embryos: Given the failure of excessPKA activity to activate Hh target genes robustly in wingdiscs it is surprising that it can activate wg and ptcexpression in embryos if this is achieved by phosphor-ylation and activation of Smo. Indeed, there is alreadysome evidence that the conventional Hh pathway is notstrongly activated by excess PKA in embryos because Fuphosphorylation, another measure of Smo activity, isonly marginally stimulated (Apionishev et al. 2005). Wetherefore investigated whether induction of wg andptc by excess PKA activity in embryos was mediated byCi binding sites. There is extensive evidence that Cibinding sites in synthetic reporter genes can confereither repression by Ci-75 or activation by Ci-155 andthat 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, ifPKA hyperactivity induces wg and ptc expression inembryos by activating Smo we would expect Ci bindingsites to be the critical feature of the Hh target genes thatallows their activation.

Expression of wg in 14 single cell-wide stripes in stage9–12 embryos is maintained by strong Hh signaling andis limited to the immediate anterior neighbors of Hh-producing cells (Ingham and McMahon 2001). This wgexpression pattern can be mimicked by a 5.1-kb regu-latory region linked to a lacZ gene (Lessing and Nusse

1998). This reporter, just like the wg gene itself, wasexpressed beyond the normal range of Hh when eitherHh or constitutively active mouse PKA catalytic subunit(mC*) was expressed ectopically in alternating seg-ments using the prd-GAL4 driver (Figure 2, B, G, andL). At the distal end of this 5.1-kb regulatory region is a1.1-kb fragment that contains several Ci binding sitesand 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). Thisreporter, Dwg-lacZ, was also expressed in wider stripesin those (alternating) segments of embryos that ex-pressed ectopic Hh, activated PKA (mC*), or a highlyexpressed transgene (Ci-T5m-s1) encoding Ci lackingkey PKA sites (Price and Kalderon 1999) (Figure 2, D,I, and N). The equivalent reporter gene (Dwg*-lacZ), inwhich the Ci binding sites have been altered by pointmutations to eliminate Ci binding in vitro, is barelyexpressed at all in wild-type embryos, demonstrating thecritical role of Ci binding sites in responding to Hh(Von Ohlen and Hooper 1997). A few embryos ex-pressed Dwg*-lacZ in response to expression of thehighly active Ci-T5m-s1 transgene in alternating seg-ments (Figure 2O). Most embryos expressing mC* inalternating segments showed no Dwg*-lacZ expression,although very weak expression in thin stripes wasdetected in a few embryos (Figure 2J). Thus, a 1.1-kbsegment of the wg enhancer is sufficient to respond toPKA hyperactivity and this response is largely contin-gent on the presence of functional Ci binding sites. Thetrace induction of Dwg*-lacZ by excess PKA likelycorresponds to residual binding of Ci to this reportersince expression of an activated form of Ci also inducedDwg*-lacZ weakly.

There is a precedent for induction of wg by Hhthrough 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 sitesbut was previously shown to repress expression of areporter gene and to confer derepression in response toHh signaling (Lessing and Nusse 1998). A reporter(WLZGc2.5L) containing two copies of box G butlacking the 1.1-kb Ci binding site region of the 5.1-kbenhancer was indeed induced ectopically by Hh, but it


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was not induced by excess PKA (Figure 2, C, H, and M).Thus, analysis of wg regulatory regions suggests that Cibinding sites are key elements required to respond toPKA hyperactivity.

Ci binding sites suffice for only a small response toexcess PKA in embryos: We then tested whether Cibinding sites suffice for induction of a reporter gene byPKA hyperactivity in stage 9–12 embryos. Barolo andPosakony (2002) established a general principle thatsignaling pathways induce artificial reporter genesefficiently if binding sites for the transcriptional effectorof the pathway are combined with binding sites for atranscriptional activator that is ubiquitous and consti-tutively active. Thus, a reporter with four Ci binding sites(4xCi-lacZ) is barely expressed in wing discs (Baroloand Posakony 2002) or in wild-type embryos; it is notresponsive to ectopic Hh and can be weakly inducedonly by expression of excess Ci from the Ci-T5m-s1transgene (data not shown). However, a reporter con-taining four Ci binding sites adjacent to three bindingsites for the transcriptional activator Grainyhead (Ci-Grh-lacZ) is expressed much like the ptc gene in wingdiscs (Barolo and Posakony 2002) and in stage 9–12embryos, in roughly single cell-wide stripes either sideof each stripe of Hh expression (Figure 3, A and D).Ectopic Hh expression in all anterior cells, achievedusing ptc-GAL4 together with UAS-Hh, expanded Ci-Grh-lacZ reporter expression to most anterior cells (Figure3C) [strong ectopic induction of wg by ectopic Hh leadsto ectopic En expression in adjacent cells, preventinginduction of Hh target genes in those En-expressing

cells (Bejsovec and Wieschaus 1993)]. However, onlya very small expansion in the domain of Ci-Grh-lacZexpression was elicited by expression of activated PKA(mC*) using ptc-GAL4 (Figure 3, B and E). This ex-pansion was much less than that observed for wg mRNAin equivalent embryos processed in parallel (Figure 3, Hand I). Thus, Ci binding sites taken out of their normalcontext in the wg enhancer respond strongly to Hh butonly very weakly to excess PKA activity.

We considered the possibility that Ci-Grh-lacZ mightbe less sensitive than wg to Hh pathway activity and thatexcess PKA might therefore activate the Hh pathway to alevel just below the threshold required to induce Ci-Grh-lacZ, leading to a deceptively small response. We testedthis possibility in two ways. First, we compared the re-sponse of Ci-Grh-lacZ to excess PKA and to PKA in-hibition. PKA inhibition induces wg mRNA less stronglythan does excess PKA activity or excess Hh (Ohlmeyer

and Kalderon 1997) (Figure 4, D–F), consistent withweak activation of the Hh pathway effector Ci. Neverthe-less, Ci-Grh-lacZ was clearly induced by PKA inhibition,contrasting with the negligible induction by excess PKAactivity (Figure 4, A–C). Thus, Ci-Grh-lacZ is clearly pro-portionally less responsive than wg to PKA hyperactivitywhen compared to either weak activation of the Hhpathway by PKA inhibition or strong activation by Hhitself.

Second, we tried to alter the threshold of the responseof Ci-Grh-lacZ by augmenting Hh pathway activity to seeif this revealed a stronger response to excess PKAactivity. We first tried to do this by coexpressing low

Figure 2.—Ci binding sites are required for induction of wg reporter genes by PKA hyperactivity in embryos. In situ RNA hy-bridization 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 expressactivated PKA catalytic subunit mC* (F–J), ectopic Hh (K–M), or ectopic activated Ci, Ci-T5m-s1 (N and O) in alternating seg-ments using prd-GAL4. Representative segments on the ventral surface (down) that express the transgenes are indicated by filledboxes aligned underneath. Expression of lacZ is from the reporter genes wg5.1-lacZ (B, G, and L), WLZ Gc2.5L (C, H, and M), Dwg-lacZ (D, I, and N), or Dwg*-lacZ (E, J, and O). Stripes of expression of wg, wg5.1-lacZ, and Dwg-lacZ (which all contain Ci-bindingsites) 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 Dwg*-lacZ (both of which lack identified strong Ci-binding sites) is not affected by mC* (H and J). WLZ Gc2.5L expression is induced byHh (M), whereas Dwg*-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.


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levels of Ci lacking PKA sites (Ci-H5m-w1), which by itselfproduces no ectopic activation of wg or Ci-Grh-lacZ(Figure 4, G and J). However, we still observed only avery small expansion of Ci-Grh-lacZ expression in alter-nating segments of embryos that expressed both Ci-H5m-w1 and mC* (Figure 4H). By contrast, Ci lackingPKA sites clearly increased the response of Ci-Grh-lacZ toPKA inhibition (Figure 4I). We then tested the con-sequences of inactivating Su(fu). Loss of Su(fu) by itselfdoes not alter the expression of either wg or Ci-Grh-lacZin 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 induceclear ectopic activation of a synthetic Hh pathway re-porter containing repeated consensus Ci binding sites.However, even in the absence of Su(fu) excess PKAinduced the Ci-Grh-lacZ reporter less strongly than didinhibition of PKA (not shown) and the Ci-Grh-lacZ re-porter was induced much less strongly than the wg geneitself (Figure 3, J and K). Thus, when tested under avariety of conditions and compared to Hh itself oractivation of Ci via PKA inhibition, excess PKA induces a

Figure 3.—Minimal inductionof a reporter gene by PKA hyper-activity through Ci binding sitesalone. In situ RNA hybridizationto stage 11–12 embryos carryinga Ci-Grh-lacZ reporter gene usinga lacZ probe (A–G) or a wg probe(H–K). (A–C) Lateral views (an-terior left, dorsal up) showingrepeated pairs of stripes of re-porter gene expression (blue)corresponding to cells either sideof thin intervening stripes of Hh-expressing cells in wild-type em-bryos (A). Ci-Grh-lacZ expressionis largely unchanged in responseto mC* expression driven by ptc-GAL4 (B) but stripes of expres-

sion are expanded in response to ectopic Hh driven by ptc-GAL4 (C). (D–K) Ventral surface showing slight, patchy broadeningof Ci-Grh-lacZ expression by mC* driven by ptc-GAL4 (E) relative to wild type (D). This broadening is considerably enhanced by lossof 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.

Figure 4.—Ci lacking PKA sites does not enhance the response of a reporter gene to excess PKA activity through Ci bindingsites. 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) inwild-type embryos (A and D) and in embryos expressing Ci lacking five PKA sites, Ci-H5m-w1 (G and J), activated PKA catalyticsubunit 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) inalternating segments using prd-GAL4. Representative segments expressing (filled boxes) and not expressing (open boxes) thetransgenes are indicated. PKA inhibition with R* in alternating segments produces a patchy expansion of the thinner anteriorCi-Grh-lacZ stripe in an anterior direction toward the stronger posterior Ci-Grh-lacZ stripe of the next segment (C). mC* causes asimilar but weaker ectopic induction of Ci-Grh-lacZ, most obvious as an alternating reduction in the unstained space between ad-jacent sets of paired stripes (B). Ci-H5m-w1 expression induces ectopic Ci-Grh-lacZ expression in the Hh-producing cells betweeneach pair of stripes in alternating segments (G) and enhances the anterior expansion of Ci-Grh-lacZ expression due to R* (I) butdoes not produce significant anterior expansions alone (G) or enhance those due to mC* expression (H). mC* induces a greaterexpansion of wg stripes than does R*, both in the absence (D–F) and presence of coexpressed Ci-H5m-w1 (J–L).


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reporter containing only Ci binding sites much lesswell than it induces wg or ptc. This suggests that excessPKA does not induce wg expression in embryos solelythrough activation of Ci.

Sequences distant from critical Ci binding sites inthe ptc gene mediate induction by excess PKA: Theability of wg and ptc genes to respond to PKA hyperac-tivity in embryos more strongly than a Ci binding sitereporter might be due to a specific local arrangement orcontext of Ci binding sites or, alternatively, to distinctadditional sequences that collaborate with Ci bindingsites to increase induction by excess PKA. For the wggene the strong response of the Dwg-lacZ reporter(Figure 2) indicates that sufficient critical sequenceslie on a 1.1-kb fragment. For ptc, a 12-kb regulatoryregion driving lacZ (ptc-lacZ) faithfully reflects thenormal regulation of the ptc gene in embryos andimaginal discs and the most proximal 800 bp of thisregion, which includes three identified Ci binding sites,suffices to direct a stripe of FE-lacZ reporter expressionat the AP border of wing discs, as seen for the endo-genous ptc transcript (Forbes et al. 1993; Alexandreet al. 1996). In embryos, ptc-lacZ and FE-lacZ are ex-pressed either side of Hh-expressing stripes and theirexpression expanded to include most anterior cellswhen Hh was ectopically expressed using ptc-GAL4(Figure 5). PKA hyperactivity strongly induced ptc-lacZexpression throughout anterior regions of each seg-ment but did not detectably affect the expressionpattern of FE-lacZ (Figure 5, B and E). The FE-lacZreporter is expressed more weakly than ptc-lacZ or Ci-Grh-lacZ reporters in response to Hh, so subtle in-duction of this reporter by excess PKA could be missed.Nevertheless, we can conclude that the clustered Ci siteswithin ptc regulatory sequences do not suffice to re-spond strongly to excess PKA activity in embryos,implying that regulatory sequences in the 11 kb up-stream of the known Ci binding sites in the ptc gene areinstrumental in responding to PKA.

Is induction of wg and ptc by PKA hyperactivity inembryos mediated by Smo phosphorylation? Theanalysis of regulatory elements described above showsthat the response of embryos to excess PKA activity iscaptured poorly by Ci binding sites alone and istherefore unlikely to be mediated solely by activationof Smo. We wished to test the role of Smo phosphory-lation in responding to PKA hyperactivity more directlyby altering the PKA sites in Smo that are known to affectSmo activity. We initially tried to do this by substitut-ing wild-type Smo with a Smo variant where the threePKA sites in question were altered to alanine residues.However, this variant lost all activity in response toboth PKA and Hh; hence we could not distinguishwhether PKA acts through Smo or separately but with arequired additional input from Smo activity (in parallel)(Apionishev et al. 2005).

To circumvent this ambiguity we tested whether aSmo variant in which the PKA sites and consensus PKA-primed and CK1-primed CK1 sites were changed toaspartate residues (SmoD1-3) (Jia et al. 2004) couldrespond to PKA and Hh in embryos. Expression ofSmoD1-3 (using prd-GAL4) in alternating segments ofembryos lacking endogenous maternal and zygotic smoactivity rescued single cell-wide stripes of wg and ptc inthe segments where SmoD1-3 was expressed, but did notinduce ectopic expression of wg or ptc beyond thenormal Hh signaling domain (Figure 6, B and F). Thus,at the levels of expression employed, SmoD1-3 does nothave detectable constitutive activity in embryos buttransduces an Hh signal efficiently. This was confirmedby coexpressing Hh (using prd-GAL4), resulting in anexpansion of wg and ptc RNA stripes to roughly theanterior limit of prd-GAL4 expression in alternatingsegments (Figure 6, D and H). Coexpression of SmoD1-3 with mC* to increase PKA activity in embryos lackingwild-type Smo produced a similar strong expansion ofwg and ptc RNA stripes in the prd-GAL4 expressionpattern (Figure 6, C and G). Thus, PKA hyperactivity can

Figure 5.—Induction of ptc re-porters by PKA hyperactivityrequires regulatory sequences be-yond the proximal Ci bindingsites. Lateral views (anterior toleft) of embryos carrying the FE-lacZ (A–C and G–I) or ptc-lacZreporter genes (D–I), showing ex-pression of lacZ (A–F) and ptcRNAs (G–I). FE-lacZ, ptc-lacZ,and ptc RNA are all expressed inthe same pattern of a pair ofstripes straddling each stripe ofHh expression in wild-type em-bryos (A, D, and G) and all stripesare expanded to include most an-terior 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 ptcand ptc-lacZ RNAs (E and H), but leaves FE-lacZ expression unaffected (B).


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induce wg and ptc strongly in the absence of critical PKAtarget sites in Smo. We therefore conclude that themajor mechanism for the induction of Hh targetgenes by excess PKA in embryos is not driven by PKAphosphorylation of Smo.

Role of Smoothened phosphorylation by PKA inwing discs: Given that excess PKA activity cannot readilyactivate the Hh pathway via Smo in embryos or wingdiscs, it is important to reconsider whether normal PKAactivity is required for Smo to respond to Hh. Sub-stitution of alanine residues at PKA and CK1 sites onSmo produces complete inactivation in wing discs (Jiaet al. 2004; Zhang et al. 2004; Apionishev et al. 2005).However, loss of activity of the major PKA catalyticsubunit PKA-C1 still allows strong Smo-dependent in-duction of the Hh-target gene collier at the AP border ofwing discs, provoking the suggestion that enzymes otherthan PKA-C1 might contribute to phosphorylation ofSmo at PKA sites (Apionishev et al. 2005). We thereforeinvestigated whether there was any requirement forSmo phosphorylation by PKA in wing discs.

There is prior evidence from assaying wing marginbristle phenotypes, and from measuring induction of asynthetic 4bs-lacZ reporter gene and anterior En expres-sion at the AP border, that PKA-C1 does contributepositively to the outcome of Hh signaling in wing discs(Jiang and Struhl 1995; Ohlmeyer and Kalderon1998; Wang and Holmgren 2000; P. Therond, per-sonal communication). We tested if this positive role ofPKA was due to phosphorylation of Smo by trying tocomplement the PKA-C1 mutant defect with SmoD1-3.Anterior PKA-C1 mutant clones induce a low levelof ectopic En cell autonomously (Ohlmeyer andKalderon 1998) but they also clearly reduced the levelof En normally induced by Hh at the AP border (Figure7A). Expression of SmoD1-3 at low levels (using C765-

GAL4 at 18�) induced some patchy ectopic anterior Enexpression but only at levels clearly lower than at the APborder. Under these conditions SmoD1-3 rescued nor-mal high levels of En at the AP border in the absence ofendogenous Smo activity (Figure 7B), showing thatSmoD1-3 activity can be increased by Hh in wing discs, asin embryos. SmoD1-3 also rescued normal levels of En atthe AP border in clones that lacked both endogenousSmo and PKA-C1 activities (Figure 7C). Thus, mimick-ing Smo phosphorylation with acidic residues elimi-nates the deficit in Hh signaling caused by loss of PKA-C1 activity. This is consistent with the idea that PKA-C1must phosphorylate Smo for Hh to signal optimally atthe AP border of wing discs.

Does Smo phosphorylation at PKA and CK1 sitesaffect all aspects of Hh signaling? Hh signaling blocksprocessing of Ci-155 to Ci-75 and also increases tran-scriptional activation by Ci-155 (Hooper and Scott2005). The activity of Ci-155 can be affected by severalpathway components, such as PKA and Cos2, that alsoaffect Ci-155 processing. However, loss of Fu kinasediminishes Ci-155 activity in response to Hh and loss ofSu(fu) can potentiate Ci-155 activity without signifi-cantly affecting Ci-155 processing (Alves et al. 1998;Ohlmeyer and Kalderon 1998). Hence, it is oftensuggested that Smo can initiate at least two distinctbiochemical pathways, one affecting Ci-155 processingand another affecting Ci-155 specific activity via Fused.Since SmoD1-3 only partially activates the Hh pathwayand can be further activated by Hh it was important totest whether mimicking Smo phosphorylation activateseach of the postulated biochemical pathways down-stream of Smo.

We found that ubiquitous expression of SmoD1-3greatly increased Ci-155 levels in anterior cells of wingdiscs, so that the normal accentuation of Ci-155 staining

Figure 6.—PKA hyperactivity strongly induceswg and ptc expression without phosphorylatingSmo. Lateral views (anterior to left) of wild-typeembryos (A and E), and embryos lacking mater-nal and zygotic smo activity (due to the smo2 allele)but expressing SmoD1-3 (B and F), SmoD1-3 andmC* (C and G), or SmoD1-3 and Hh (D and H)in alternating segments using prd-GAL4. Expres-sion of wg is rescued by SmoD1-3 in alternatingstripes of normal width (B). These rescued stripesare greatly expanded by coexpression of mC* (C)or Hh (D). Similarly, pairs of ptc stripes are res-cued in alternating segments by SmoD1-3 (F)and expanded by coexpression of mC* (G) orHh (H).


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at the AP border was no longer discernible (Figure 8, Aand C). This is consistent with inhibition of Ci-155processing to Ci-75. SmoD1-3 expression also increasedFu phosphorylation substantially, as measured in ex-tracts of wing discs (Figure 8B), as observed previouslyfor a similar Smo variant in cultured cells (Zhang et al.2004). Hyperphosphorylation of Fu also occurs in cellsresponding to Hh and may reflect activation of Fu(Hooper and Scott 2005). More importantly, in wingdiscs that lack Fu kinase activity the strong induction ofptc-lacZ in anterior cells by SmoD1-3 (using C765-GAL4at 25�) was greatly curtailed and ectopic induction of Enby SmoD1-3 was completely eliminated (Figure 8E).Thus, SmoD1-3 must constitutively activate the branchof 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 fromexperiments in which SmoD1-3 was ubiquitously ex-pressed 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 Ciphosphorylation, but did not significantly reduce ec-topic anterior expression of either ptc-lacZ or En (Figure8, C and D). Thus, it appears that two arguably separateaspects of Hh signaling are phenocopied by changingSmo PKA and CK1 sites to acidic residues.

DISCUSSION

When the role of PKA in Hh signaling was firstdiscovered it appeared that PKA acted simply to silencethe pathway in the absence of Hh (Jiang and Struhl

1995; Lepage et al. 1995; Li et al. 1995; Pan and Rubin1995). This aspect of PKA function has been studiedfurther, revealing that it is conserved in vertebrate Hhsignaling and can be explained adequately by thephosphorylation of three clustered consensus PKA siteson Ci-155 (Jia et al. 2005; Pan et al. 2006; Smelkinsonand Kalderon 2006; Wang and Li 2006). Loss of thesesites, loss of PKA activity, and even the consequences ofexcessive PKA activity in wing discs all lead to a coherentpicture of how PKA silences Ci and the Hh signalingpathway in the absence of Hh. This role of PKA haddisguised recognition of any potential positive role forPKA in transduction of an Hh signal on the basis ofsimply manipulating PKA activity. Indeed, a positive rolefor PKA in Hh signaling was clearly revealed only byaltering PKA (and PKA-primed CK1) phosphorylationsites in Smo; changes to alanine residues eliminatedactivity and changes to acidic residues endowed someconstitutive activity ( Jia et al. 2004; Zhang et al. 2004;Apionishev et al. 2005). Those and other studies leftopen a number of significant questions. Are the con-sensus PKA sites on Smo actually phosphorylated byPKA and only by PKA, and is phosphorylation of Smo byPKA required to transmit an Hh signal? Does Smo withacidic residues at PKA and CK1 sites mimic the con-sequences of phosphorylation at those sites, and does itelicit 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 PKAin 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.

Figure 7.—SmoD1-3 restoresEn expression to PKA mutantclones at the AP border. (A–C)Third instar wing discs (anteriorleft, dorsal up) stained for En pro-tein (red), GFP (green) to markhomozygous mutant clones lack-ing GFP, and ptc-lacZ expressionusing antibodies to b-galactosi-dase (blue). En expression atthe AP border is substantially lostin PKA-C1 mutant clones (arrow-head, A) but is restored to bothsmo mutant clones (arrowhead,B) and smo PKA-C1 double mu-tant clones (arrowhead, C) atthe AP border by expressionof UAS-SmoD1-3 driven by C765-GAL4 at 18�. In each case anteriorEn expression is distinguishedfrom posterior En expression byits overlap with ptc-lacZ expres-sion, which is strictly confined toanterior cells. White lines markthe posterior limit of ptc-lacZ ex-

pression. 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.


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2005). Hence, Smo PKA sites must be critical in theirphosphorylated form and elimination of the relevantprotein kinase activity should prevent all responses toHh. Expression of a dominant-negative PKA regulatorysubunit (R*) in embryos does substantially reduce Fuphosphorylation induced by endogenous or ectopicallyexpressed Hh, consistent with the idea that PKA is themajor protein kinase that phosphorylates Smo on PKAsites in embryos (Apionishev et al. 2005). However, PKAinhibition with R* in embryos does not prevent allHh-stimulated phosphorylation of Fu or Hh-dependentmaintenance of wg expression (Ohlmeyer andKalderon 1997; Apionishev et al. 2005). Since PKAinhibition by R* is likely incomplete it is not possible todistinguish whether these residual responses to Hhresult from phosphorylation of Smo by residual PKAactivity or by another protein kinase, but it should benoted that PKA inhibition by R* is sufficient to producevery high levels of Ci-155, indicative of a complete blockin Ci-155 processing (Lane and Kalderon 1993; Laneand Kalderon 1994; Ohlmeyer and Kalderon 1997;Apionishev et al. 2005).

In wing discs PKA-C1 activity can be eliminatedcleanly 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 withdemonstrated developmental functions, even though atleast one other gene encodes an equivalent biochemicalactivity (Lane and Kalderon 1993; Melendez et al.1995). Loss of PKA-C1 activity in wing disc clones doesreduce Hh signaling, as revealed most clearly by stronglyreduced 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 mutantclones at the AP border can be complemented byexpressing SmoD1-3 in place of wild-type Smo. Thissupports the idea that PKA-C1 must phosphorylateSmo for Hh to elicit maximal pathway activity, whichis required for strong induction of En. It is not sostraightforward to determine whether Hh requiresPKA-C1 activity to induce target genes such as collier(col) or ptc, which require lower levels of Hh pathwayactivity. This is because loss of PKA-C1 by itself inducesstrong ectopic ptc and col expression. Nevertheless,when induction of col in PKA-C1 mutant clones waslargely suppressed by reducing the dose of ci, it was clearthat Hh still induced high levels of col in PKA-C1 mutantclones at the AP border and that this induction requiredSmo activity (Apionishev et al. 2005). Thus, Smo retainssome but not maximal activity in response to Hh when

Figure 8.—SmoD1-3 stabilizes Ci-155, pro-motes Fu phosphorylation, and requires Fu ki-nase activity to activate Ci strongly. (A, C–E)Expression of full-length Ci-155 detected by anti-body 2A1 (red, first column), ptc-lacZ (green, sec-ond column), and En protein (green, thirdcolumn) in wild-type discs (A) and in discs ex-pressing SmoD1-3 alone (C), SmoD1-3 togetherwith activated PKA mC* (D), and SmoD1-3 inthe absence of Fu kinase activity (E), usingC765-GAL4 at 25�. Ci-155 is selectively stabilizedat the AP border of wild-type wing discs (A) butis additionally stabilized in all anterior cells bySmoD1-3 (C). Ci-155 levels induced by SmoD1-3 are greatly reduced by excess PKA without re-ducing ectopic anterior ptc-lacZ or En inductionsignificantly (D). ptc-lacZ and En induction bySmoD1-3 are drastically reduced in discs hemizy-gous for the fumH63 allele, which encodes kinase-deficient Fu (E). (B) Western blot using antibodyto Fu of extracts of wing discs expressing Ci-H5m-w1 (as a control), SmoD1-3, activated PKA(mC*), or mC* together with CK1 (Dbt) usingC765-GAL4 at 25�. Only SmoD1-3 strongly indu-ces a change of mobility of Fu characteristic ofthe hyperphosphorylated species (Fu-p).


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PKA-C1 activity is lost, implying that another kinase canphosphorylate Smo at PKA sites in wing discs. Thisinference is also supported by the observations that Smois stabilized in anterior cells when its PKA sites aresubstituted 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 samemanipulations of PKA-C1 completely block processingof Ci-155 to Ci-75 and strongly activate Ci-155 in wingdiscs (Methot and Basler 2000; Hooper and Scott2005). Why might Smo and Ci-155 show differentsensitivities to PKA-C1? One possibility is that scaffold-ing molecules may allow special access of PKA-C1 to Ci-155 that is not available to other kinases that mightotherwise phosphorylate PKA sites. Indeed, Cos2 doesappear to ensure efficient phosphorylation of Ci-155 byPKA-C1 by binding to both components (Zhang et al.2005). However, Cos2 also binds to Smo (Hooper andScott 2005) and therefore presumably also providessimilarly enhanced access for PKA-C1. A more likelyexplanation of the different responses of Smo and Ci-155 to PKA-C1 manipulation concerns the stoichiome-try of phosphorylation. A key functional consequence ofCi-155 phosphorylation is the binding of Slimb, and thisrequires extensive phosphorylation of Ci-155 primedby each of the three relevant PKA sites ( Jia et al. 2005;Smelkinson and Kalderon 2006). Thus, any signifi-cant reduction in the rate of phosphorylation of thesesites might be translated into strong stabilization of Ci-155. Conversely, since Smo retains considerable activityin the absence of PKA-C1 we speculate that a low rate ofphosphorylation of Smo at PKA sites may suffice for it tobe active.

Is phosphorylation of Smo at PKA and CK1 sitessufficient to activate the Hh pathway? The discoverythat substitution of multiple PKA and CK1 site Serines ofSmo with acidic residues conferred constitutive activityprovoked the simple hypothesis that activation of Smoby Hh can be attributed largely to an Hh-stimulatedincrease 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 acidicresidues at PKA and CK1 sites (SmoD1-3) and of theconsequences of forced phosphorylation of Smo do notsupport this simple hypothesis.

First, we found that Hh can increase pathway activity incells expressing SmoD1-3. This effect is small in wing discs,where (overexpressed) SmoD1-3 has strong constitutiveactivity and was described previously (Jia et al. 2004). How-ever, in embryos SmoD1-3 exhibited no clear constitutiveactivity but transduced a normal response to Hh. Thus,Hh must elicit changes in Smo activity other thanphosphorylation at PKA and CK1 sites that are sufficientlyimportant to convert pathway activity from a silent state tobeing fully active in embryos. We speculate that these

(unknown) changes are conserved elements of all Hhsignaling pathways and that phosphorylation of Drosoph-ila Smo at PKA and CK1 sites, which are not conserved invertebrate Smo proteins, is a prerequisite for DrosophilaSmo to undergo these Hh-dependent changes.

Second, we found that excess PKA activity and CK1activity cannot reproduce the ectopic activation of Hhtarget genes induced by expression of SmoD1-3 (Figure1 and supplemental Figure S1 at http://www.genetics.org/supplemental/). This was true despite our attemptsto sensitize Hh target gene induction by eliminatingSu(fu) or by providing additional processing-resistantCi-155. An analogous difference in the potency ofSmoD1-3 and excess PKA and CK1 activity was observedwhen using Fu phosphorylation as a measure of Hhpathway activity in wing discs (Figure 8).

Why are excess PKA and CK1 activities not sufficientto activate Smo? One possibility is that overexpression ofPKA or CK1 did not effectively stimulate Smo phos-phorylation. We do not favor this explanation becauseboth of the protein kinases used are thought to associatewith Cos2 (Zhang et al. 2005) and therefore should havegood access to Smo, and analogous overexpression studiesshow that each can lower Ci-155 levels at the AP border,implying that they induce significant changes in Ci-155phosphorylation (Price and Kalderon 2002).

Another possibility is that PKA or CK1 may have tar-gets other than Smo that reduce Hh signaling pathwayactivity, obscuring the effects of any potential activationmediated by Smo phosphorylation. Ci-155 is certainlyone such target but we excluded this confoundinginfluence by coexpression of a Ci mutant lacking allknown regulatory PKA sites and also by measuring Fuphosphorylation in addition to Hh target gene activa-tion. It is conceivable that there are additional in-hibitory targets for PKA in the Hh pathway because weobserved that the induction of ptc-lacZ in posterior wingdisc cells by a PKA-resistant Ci variant (Ci-H5m) was,surprisingly, reduced by excess PKA activity.

Finally, our favored explanation is that Smo withacidic residues at PKA and CK1 sites behaves signifi-cantly differently from Smo that is phosphorylated atthose sites. We have previously argued that phosphory-lation is essential for the activity of Smo in the presenceof Hh but also targets Smo for degradation in theabsence of Hh (Apionishev et al. 2005). We further spec-ulate that Hh might normally stabilize the phosphory-lated state of Smo rather than actively promoting Smophosphorylation and that acidic residues might mimicSmo activation by phosphorylation without simulta-neously promoting Smo degradation in the absence ofHh. In this scenario SmoD1-3 would accumulate andexhibit constitutive activity, especially when overex-pressed, but it would not be possible to accumulateactivated Smo very effectively in the absence of Hh by in-creasing only its rate of phosphorylation at PKA and CK1sites. The hypothesis that Hh stabilizes phosphorylated


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Smo rather than promoting Smo phosphorylation isalso consistent with the earlier conjecture that Smoactivation by Hh requires only a low rate of phos-phorylation at PKA sites.

A significant question for the future is how phosphor-ylation of Smo contributes to its activity. We have someclues from examining the properties of SmoD1-3 inwing discs. SmoD1-3 stabilizes Ci-155, induces phos-phorylation of Fu, shows substantial dependence on Fukinase activity for induction of Hh target genes and cansuffice for strong induction of anterior En expression inwing discs. These results suggest that SmoD1-3 activatestwo genetically separable aspects of Hh signaling (Ci-155 stabilization and the Fu kinase signaling pathway)that are sometimes hypothesized to correspond to twobiochemically distinct pathways (Ogden et al. 2004;Hooper and Scott 2005). The nonphysiological cir-cumstances of using high levels of expression and acidicresidues in place of phosphorylation may contribute toone or the other of the apparent dual attributes ofSmoD1-3 in Hh signaling. Nevertheless, it appears thatphosphorylation of Smo at PKA and CK1 sites at leastmakes Smo competent to activate each known aspect ofthe Hh signaling pathway. This fits with the idea thatSmo phosphorylation may be constitutive but necessaryto make Smo competent to respond to Hh.

Induction of Hh target genes in embryos by factorsother than Ci: We found that strong ectopic activationof the Hh target genes, wg and ptc, by excess PKA activityin embryos is the consequence of two distinguishableresponses. First, PKA does appear to induce target genesthrough Ci binding sites, consistent with enhancingSmo activity through phosphorylation. However, thisresponse alone would result in only a very small induc-tion of Hh target genes. The salient evidence is that PKAhyperactivity induces (i) detectable, but very limited, ec-topic expression of a reporter gene that essentially con-tains only Ci binding sites (Figures 3 and 4), (ii) clearectopic expression of a wg reporter gene that dependson the presence of Ci binding sites (Figure 1), and (iii) asmall increase in Fu phosphorylation (Apionishev et al.2005). Second, PKA hyperactivity induces wg and ptctranscription principally through regulatory elementsother than Ci binding sites and through a mechanismthat does not require a change in phosphorylation atSmo PKA sites. The salient evidence is that the responseto excess PKA is greatly enhanced if regulatory elementsfrom the wg and ptc genes other than just Ci bindingsites are present (Figures 2–5) and that wg and ptc arestrongly induced by excess PKA activity even when theonly Smo protein present has acidic residue substituentsat PKA and CK1 sites (Figure 6).

The dual consequences of excess PKA described aboveclarify a potential misconception in the literature thatPKA can strongly activate the Hh pathway through Smoand substantiate the idea that excess PKA produces onlya small activation of the Hh pathway through phosphor-

ylation of Smo, whether assayed in wing discs or em-bryos. These results also raise the question of the natureand physiological significance of the pathway thatconnects excess PKA activity to induction of wg and ptcthrough enhancer elements other than Ci binding sites.

PKA is known to phosphorylate many proteins thatcan influence transcription (Conkright et al. 2003;Rochette-Egly 2003; Martin et al. 2004; Poels andVanden Broeck 2004) and thus its ability to activate wgand ptc through sites other than Ci binding sites whenhyperactive may simply be an artifact of this nonphysio-logical condition An alternative possibility is that thisconsequence of excess PKA activity exposes a regulatorymechanism that is relevant to target gene activation byHh in embryos. There is some evidence for transcrip-tion factors other than Ci contributing to induction ofHh target genes in embryos (Lessing and Nusse 1998;Gallet et al. 2000; Muller and Basler 2000). Further-more, it is clear that there must be interactions betweenCi and other gene-specific transcription factors thatunderlie both the different sensitivity of genes withequivalent Ci binding sites to activation by Ci-155 andrepression by Ci-75 and the tissue-specific responses ofmost genes to Hh (Muller and Basler 2000; Hooper

and Scott 2005). Whether Hh signaling affects theactivity or interactions of transcription factors thatcollaborate with Ci is not presently known.

An intriguing aspect of the ectopic induction of wgand ptc by excess PKA through sites other than Cibinding sites is its dependence on concomitant activa-tion through Ci binding sites. Thus, induction of wg andptc by excess PKA requires both Smo and Ci activities(Ohlmeyer and Kalderon 1997) and requires func-tional Ci binding sites within the Dwg-lacZ reporter gene(Figure 2). Even the PKA sites on Smo are required forwg to respond to excess PKA (Apionishev et al. 2005),consistent with the idea that some activation of Smo isrequired. We do not yet, however, have any indicationthat Hh signaling normally involves the PKA-responsiveregions of wg and ptc enhancers that can collaboratewith Ci binding sites. Indeed, both Ci-Grh-lacZ and FE-lacZ reporters, which lack key regulatory regions re-quired for a strong response to excess PKA activity, areclearly induced by Hh. There are, however, caveats tothis evidence; induction of Ci-Grh-lacZ depends on thesynthetic Grh binding sites as well as its Ci binding sites(Barolo and Posakony 2002) and the FE-lacZ reporteris 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 trans-mitted largely through Ci and supplemented by con-tributions from enhancer elements other than Cibinding sites, including those that are responsive toPKA. One pathway that is known to supplement Hh-induced wg expression in embryos is the Wg auto-regulation pathway (Hooper 1994; Yoffe et al. 1995).However, this does not appear to be relevant to the


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PKA-responsive elements under discussion here be-cause PKA hyperactivity did not substitute for therequirement for Wg activity to maintain stripes of wgexpression (data not shown) and PKA hyperactivity alsoinduces ectopic ptc expression, which does not dependon Wg activity for its expression (data not shown). In thefuture, the clearest way to test the significance for Hhsignaling of regulatory elements responsive to excessPKA will be to define and then alter those regulatoryelements.

We thank Derek Lessing, Roel Nusse, Joan Hooper, Scott Barolo,David Robbins, Jim Posakony, Jianhang Jia, Jin Jiang, Johanna Ohl-meyer, and Mary Ann Price for providing key reagents. This work wassupported by grant GM-41815 from the National Institutes of Health.

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