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Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated byHedgehogLaurent Ruel, Ralph Rodriguez, Armel Gallet, Laurence Lavenant-Staccini and Pascal P. Thérond

The mechanisms involved in transduction of the Hedgehog(Hh) signal are of considerable interest to developmental andcancer biologists. Stabilization of the integral membraneprotein Smoothened (Smo) at the plasma membrane is acrucial step in Hh signalling but the molecular eventsimmediately downstream of Smo remain to be elucidated. Wehave shown previously that the transcriptional mediatorCubitus interruptus (Ci) is associated in a protein complex withat least two other proteins, the kinesin-like Costal2 (Cos2) andthe serine–threonine kinase Fused (Fu). This protein complexgoverns the access of Ci to the nucleus. Here we show that,consequent on the stabilization of Smo, Cos2 and Fu aredestabilized. Moreover, we find that the Cos2–Fu–Ci proteincomplex is associated with Smo in membrane fractions both invitro and in vivo. We also show that Cos2 binding on Smo isnecessary for the Hh-dependent dissociation of Ci from thiscomplex. We propose that the association of the Cos2 proteincomplex with Smo at the plasma membrane controls thestability of the complex and allows Ci activation, eliciting itsnuclear translocation.

The hh gene family encode secreted proteins that control cell division,growth and patterning during several developmental processes in bothvertebrates and invertebrates1. During Drosophila embryonic and limbdevelopment, posterior compartment (P) cells express the proteinEngrailed (En), which induces hh expression2,3. In the anterior com-partment (A) cells, Hh activates target genes including the decapenta-plegic (leg and wing only) and wingless (embryo and leg only)morphogens as well as the Hh receptor patched (ptc)1. In P cells, how-ever, the absence of the transcriptional mediator Ci means that Hh tar-get genes are not activated in the Hh-expressing domain4.

Two integral membrane proteins are involved in Hh signal recep-tion. Ptc binds to Hh, whereas the seven-transmembrane protein Smotransduces the signal5–8. Genetic analysis has shown that Ptc activityantagonizes Hh signalling by blocking Smo activity9. Hh binding toPtc might trigger the internalization and degradation of Ptc, whileinducing the stabilization of Smo at the plasma membrane10,11. As a

result of Smo activation, the transcription factor Ci is activated andtranslocated into the nucleus. However, the means by which the signalis transmitted from Smo to Ci is not known. We have previouslyshown that Ci and Cos2 form a multiprotein complex that alsoincludes the Fu kinase12,13. In the absence of Hh, Cos2 binds Cidirectly and maintains it outside the nucleus14. Despite this wealth ofinformation, the signalling link between Smo and the Cos2–Fu–Cicomplex remains elusive. Here we explore the possibility that Smomight transmit the signal by direct physical contact with the Cos2complex.

First we analysed the distribution of Smo, Cos2 and Fu in embry-onic ectodermal and imaginal wing disc cells. Smo protein is destabi-lized in A cells that do not receive Hh (Fig. 1b–d, f, i, h, k) but isstabilized both in A cells at the anteroposterior (A/P) border and in Pcells that receive a high concentration of Hh. This stabilizationdepends on the absence of ptc expression in P cells and on Hh-dependent Ptc inhibition in A cells at the A/P border (data not shown;refs 10, 15, 16). Conversely, we found that Cos2 and Fu levels arehigher in A cells than in P cells in both embryonic segments and larvalimaginal discs (Fig. 1a, c, e–g, i, j, and Supplementary Information,Fig. S1). Nevertheless, A cells at the A/P border showed a lower con-centration of Cos2 and Fu with a sharper contrast in embryonic cellscompared with a gradual decrease in disc cells. This is in contrast tothe uniform distribution of the corresponding mRNA13,17, indicatinga possible post-transcriptional regulation of both Cos2 and Fu by theHh signal.

To test this hypothesis, we monitored Cos2 and Fu protein levels inthe wing imaginal disc in clones of cells from three different geneticbackgrounds in which ectopic Hh signalling is activated9,18–20. First,Cos2 and Fu levels were decreased in clones of cells ectopicallyexpressing Hh in the anterior compartment (Fig. 1l, m, andSupplementary Information, Fig. S1e, f). Activation of Hh targetgenes can also be induced by enhancing Smo stabilization after ectopi-cally expressing Smo (Fig. 1n, and data not shown). Clones of A cellsexpressing Smo induced Cos2 (Fig. 1o, p) and Fu destabilization(Supplementary Information, Fig. S1i, j). Finally, the same effect wasobserved in clones of A cells mutant for ptc (Fig. 1q, r, and

Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 02, France.Correspondence should be addressed to P.P.T. (e-mail: therond@unice.fr).

Published online: 1 October 2003; DOI: 10.1038/ncb1052

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Supplementary Information, Fig. S1g, h) in which Hh target genesare ectopically expressed9. We proposed that the high levels of Cos2and Fu in A cells constitute the default state because inhibition of Hh

signalling in smo mutant clones induced an increase in both Cos2(Fig. 1s, t) and Fu (Supplementary Information Fig. S1k, l) in the poste-rior compartment. These data indicate that the apparent destabilizationof Cos2 and Fu in P cells and in A cells at the A/P border might be a directconsequence of Smo stabilization. Regulation of Cos2, Fu and Smo sta-bility can be detected only in Hh-expressing cells or in cells very close tothe Hh source. This suggests that this process is consequent on high acti-vation of the Hh pathway or is detectable only under these conditions.

We performed a biochemical analysis of the effects of Hh signallingon Cos2 and Fu stability in cultured cl-8 imaginal disc cells, which areresponsive to Hh. Upon induction of Hh signalling, phosphorylation ofboth Fu and Cos2 was observed (Fig. 2a; refs 12, 21). The appearance ofthese phosphorylated isoforms was accompanied by a decrease in Cos2and Fu abundance (70% decrease after 2 h of induction). In the sametime course, an increase in Smo concentration was also observed, asdescribed previously (Fig. 2a)10. Thus, the stabilities of Smo andCos2–Fu seem to be mutually exclusive in imaginal discs and inembryos (Fig. 1). Furthermore, the kinetics of Cos2 and Fu destabiliza-tion is correlated with the time course of Smo stabilization (Fig. 2a),which has been shown to be independent of translation regulation10.

Taken together, these data suggest that Hh signalling induces thedestabilization of both Cos2 and Fu, which is mechanistically relatedto Smo stabilization. Because the level of overexpressed Smo (Fig. 1n)is beyond the physiological range we could not assess whether a changeof Smo level is sufficient for pathway activation. Regulation of Cos2and Fu stability occurs at the level of Smo or downstream of it butupstream of transcriptional regulation by Ci because it also occurs in Pcells that do not express Ci. Nevertheless, both Cos2 and Fu proteinswere still present in imaginal disc P cells (Fig. 1) and also after pro-longed treatment of cultured cells with Hh (data not shown), indicat-ing that the Smo-dependent destabilization of Cos2 and Fu did nottrigger a complete disappearance of these two proteins.

To confirm the epistatic relationship between Smo, Ptc, Fu andCos2, we used a combination of a Hh cell culture assay21–23 and RNAinterference (RNAi)-mediated disruption of gene function24.Treatment of Schneider (S2) cells with Hh-conditioned mediuminduced phosphorylation of Smo (ref. 10), Cos2 and Fu (Fig. 2b)12.This treatment also induced stabilization and activation of Ci (data notshown)22, resulting in a fourfold activation of a luciferase reportergene construct (Fig. 2b)23.

RNAi targeting of Smo inhibited the response to Hh, correlated withan increase in Cos2 and Fu protein abundance but a decrease in theirphosphorylation (Fig. 2b). This confirms that Smo activity controlsboth the phosphorylation and stability of Cos2 and Fu. In contrast,RNAi targeting of Cos2 or Ptc resulted in either constitutive activationof the pathway or enhanced responsiveness to Hh (Fig. 2b). We alsonoted that Cos2 is necessary for Fu stability both in vitro and in vivo(Fig. 2b, and data not shown).

To performed epistatic analysis, double-stranded RNA (dsRNA)species were combined in transfection experiments. Constitutive activa-tion of Hh signalling induced by Ptc RNAi was inhibited by Smo inacti-vation. Conversely, constitutive activation of Hh signalling induced byCos2 RNAi was unaffected by Smo inactivation. These epistatic relation-ships place Cos2 function downstream of Ptc and Smo.

We confirmed this analysis in vivo by showing that Smo levels wereunaffected in cos2 mutant A or P cells (Fig. 2c, d). In contrast, Smo sta-bility was decreased in Cos2-overexpressing clones in the P compart-ment (Fig. 2e, f), indicating a negative feedback loop by which Cos2represses the stability of Smo. This raised the possibility that Cos2might interact with Smo, consequently interfering with the Smo stabi-lization process.

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Figure 1 Destabilization of Cos2 depends on Hh signalling. All pictures areof wing imaginal discs of third-instar larvae except a–e, which are of wild-type embryos at stage 10. (a, b) P cells in embryos were labelled by En(green) (a) and by Hh (red) (b). (c–e) Magnification of two abdominalsegments showing the Cos2 (red) and Smo (green) stabilization domains,which are mutually exclusive (bracketed arrows mark the hh-expressing andhh-receiving cells). (f–k) Wild-type discs. Cos2 is stabilized in most Acompartments but not in A/P border cells labelled for Ptc in green (f–i). Smo(blue) is stabilized in the P compartment and in A cells at the A/P border(f, h, i, k). (i–k) Z-sections of discs with apical position towards the top(arrows mark the limit of Cos2 and Smo stability). Antibodies recognizingdifferent domains of Cos2 gave similar results (data not shown). (l, m) Flp-out anterior clones of hh-expressing cells (marked by GFP) induceddestabilization of Cos2 (red). (n) Smo overexpression in the wing pouchusing the MS1096 driver induced Smo stabilization (red) and dppexpression (green) in the anterior compartment. (o–r) Cos2 is destabilizedboth in Flp-out anterior clones of smo-expressing cells (o, p) and in anteriorclones of cells homozygous mutant for ptc (q, r). (s, t) Cos2 is stabilized inposterior clones of cells mutant for smo. Arrowheads indicate clonesrevealed by an absence of β-Gal (green in q) or Myc (green in s), or by thepresence of GFP (green in l and o). The A/P border is indicated by an arrow.

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We investigated this hypothesis by analysing whether Cos2 and Smoare present in the same membrane fractions. cl-8 cell membrane andcytosolic fractions were obtained by differential centrifugation of celllysates25. Smo protein was present in vesicle fractions (V1 and V2 inFig. 3a) and enriched in the Neurotactin (Nrt)-containing plasmamembrane fraction (V0). Interestingly, Cos2, Fu and Ci were also pres-ent in vesicle fractions. Cos2 and Fu immunoreactivity was more con-centrated in vesicle fractions V1 and V2. This enrichment in vesiclelocalization was disrupted when the cells were treated with Hh (Fig.3a). This indicates that an intracellular vesicular pool of Cos2 and Fumight be destabilized after the activation of Hh signalling. To confirmour biochemical findings, we investigated the subcellular localizationof these proteins in vivo.

Embryonic ectodermal cells were chosen because they are 3–4-fold thesize of imaginal disc cells and are therefore more suitable for subcellular

analyses. Detailed confocal analysis showed that Cos2 is present incytosolic punctate structures that might represent a vesicular localiza-tion (Fig. 3d). Moreover, Cos2 is also present at the plasma membrane(labelled with Nrt26). Similar staining was observed for Fu (data notshown). Furthermore, Smo was also present in cytosolic punctatestructures but was strongly stabilized at the plasma membrane of Hh-expressing and receiving cells (Fig. 3c). In these cells Cos2 cytosolicstaining is weaker. Importantly, double labelling with Cos2 and Smoshowed that most plasma membrane-bound Cos2 colocalized withSmo (Fig. 3e) and was slightly enriched in Hh-receiving cells. In con-trol experiments we did not find any colocalization between Smo andvesicular Rab7, which also showed a punctate staining (Fig. 3i–k).

To confirm that Cos2, Fu and Smo are present in the same mem-brane-bound protein complex we analysed their interaction in cul-tured cl-8 cells. Smo co-immunoprecipitated with Cos2 and Fu

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As a control we showed that RNAi of Smo blocked Hh signallingconstitutively induced by RNAi of Ptc (lanes 11 and 12). Bottom: westernblot analysis shows phosphorylation and stabilization of both Cos2 and Fu.Fu is destabilized in Cos2 RNAi. Immunoprecipitation followed by westernblot for Smo confirms Smo stabilization when cells are activated by Hh ortreated by Ptc RNAi. A western blot for α-tubulin is shown as a control forprotein loading. (c–f) Wing imaginal discs of third instar larvae. In clones ofcells homozygous mutant for cos2 (marked by the absence of β-Gal in green)Smo (in red) is not affected (c, d). In Flp-out clones of cos2-expressing cells(marked by GFP) Smo is destabilized (e, f).

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(Fig. 3b). Similarly, we also detected Smo and Fu in Cos2 immunopre-cipitates. Ptc was not present in the Cos2 immune complex (data notshown). In Hh treated cells the association of Smo with Cos2 and Fuproteins was enriched in Cos2 immunoprecipitates (Fig. 3b), confirm-ing our observation in vivo.

The role of the Cos2–Fu complex is to retain Ci out of thenucleus12,14. Could Ci therefore be present in the same protein com-plex with Smo? Extensive colocalization of Ci with Cos2 in punctatestructures was found both at the plasma membrane and in the cytosolof cells not receiving Hh (Fig. 3f–h). This was corroborated by the

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Figure 3 Cos2, Fu and Ci form a protein complex with Smo. (a) Westernblot analysis shows that endogenous Ci, Cos2, Fu and Smo are present invesicle fractions prepared by differential centrifugation (V0, 40,000gmax;V1, 120,000gmax; V2, 240,000gmax). The plasma membrane marker Nrtand cytosolic α-tubulin are enriched in the V0 and S3 cytosolic fractionsrespectively. (b) Right: in both Cos2 or Smo immunoprecipitation (IP),endogenous Cos2, Fu, Ci and Smo co-precipitated. Ci is dissociated fromthe protein complex in Hh-treated cells. Left: western blot (WB) of cellextracts before immunoprecipitation. (c–h) Confocal x/y sub-apical sections(magnification ×2,000) of wild-type stage 11 embryos shown in the leftpanels. Double-headed arrows delimit the Hh-expressing and receiving

cells; stars indicate the Hh-receiving cells. Cos2 (d, g) is in red. Smo (c, i)and Ci (f) are in green. (e, h) Overlaps (yellow) of Cos2 with Smo (e) and ofCos2 with Ci (h) stainings superimposed on the Nrt staining (blue).Cytosolic Cos2 diminished, whereas Smo was stabilized at the plasmamembrane in Hh-expressing/receiving cells (c, d). Cos2 and Smocolocalized at plasma membrane with a slight increase in Hh-expressing/receiving cells (e). Cos2 and Ci colocalized in the cytoplasm andat the plasma membrane of non-Hh-receiving cells (h). This colocalizationdecreased about sixfold in Hh-receiving cells. For technical reasons wecould not assess Ci and Smo colocalization. (i–k) Smo did not colocalizewith vesicular Rab7 (red).

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presence of Ci in the Smo–Cos2–Fu immune complex (Fig. 3b).Interestingly, although Ci was stabilized in Hh-receiving cells, muchless colocalization was detected between Ci and Cos2, suggesting a

dissociation of Ci from Cos2 when it is associated with Smo at the plasmamembrane (Fig. 3f–h). Furthermore, after the activation of Hh signallingin cl-8 cells, Ci was absent from the Cos2 and Smo immunoprecipitates.

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Figure 4 Cos2 binding on Smo is necessary for the Hh-dependentregulation of Ci. (a) A Smo-HA construct was co-transfected in S2 cellswith various Cos2–Myc constructs. (b) After Smo immunoprecipitation(IP), Smo and Cos2 were detected with anti-HA (top panel) or anti-Myc(middle panel) antibodies respectively. The arrow in the middle panelpoints to the smallest Cos2–Myc construct that co-immunoprecipitatedwith Smo. Lower panel, Cos2–Myc constructs before immunoprecipitation.Similar results were obtained when Cos2–Myc constructs were used as abait to precipitate Smo (data not shown). WB, western blot. (c)Transfection of Ci gave a constitutive pathway activation (first measure onthe left). Hh-N treatment induced a 3–4-fold activation of the reportergene activity. Ci-dependent constitutive activation of the pathway isrepressed by 80% by wild-type Cos2 (1–1201) and Cos2 (1–900) but not

by Cos2 (900–1201). Repression of Cos2 (1–1201), but not that of Cos2(1–900), is released by Hh signal. Cos2 (900–1201) blocked Hh-dependent activation of the reporter gene. (d) Cos2 (900–1201) competeswith endogenous Cos2 for Smo-binding sites. After immunoprecipitation,Smo was detected with anti-HA antibody (top panel). Endogenous andtransfected Cos2 proteins were detected with anti-Cos2 (second panel) oranti-Myc antibodies (third panel) respectively. The endogenous Smo–Cos2association is disrupted when Cos2–Myc (900–1201) or Cos2–Myc(1–1201) are present (compare lanes 2 and 3 with lane 5 in the secondpanel). Cos2–Myc (1–900) does not compete with endogenous Cos2.Bottom panel, Cos2–Myc proteins in the initial lysate. The epitopesrecognized by anti-Cos2 antibody (509–751) do not permit the detectionof Cos2–Myc (900–1201).

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This strongly suggests a Hh-dependent dissociation of Ci from theSmo–Fu–Cos2 complex. Nevertheless, in Hh-receiving cells Ci is stillpresent in vesicular fractions and punctate structures (Fig. 3a, f),indicating that the release of Ci from the membrane might be amulti-step process.

Finally, by using Cos2 deletion mapping we have identified the Smo-binding domain on Cos2 (Fig. 4a, b). The last 301 amino acids of theCos2 carboxy terminus are sufficient to provide full association withSmo. This domain corresponds to a putative cargo domain of the pro-tein13 and is different from the Ci-binding domain on Cos2 (residues348–546)27. The inhibitory effect of Cos2 on Ci is suppressed uponactivation of Hh signalling22. We reasoned that if the association ofCos2 with Smo is necessary for the release of Ci from Cos2, then a Cos2protein lacking the Smo-binding domain but not the Ci-bindingdomain should not be sensitive to Hh regulation. Constitutive Ci activ-ity is repressed in a similar manner with Cos2 wild-type protein (Cos2(1–1201)) or with Cos2 protein lacking the Smo-binding site (Cos2(1–900); Fig. 4c). Repression by Cos2 (1–900) was not inhibited by Hhsignalling, whereas Hh signal induced a fourfold activation in the pres-ence of Cos2 (1–1201) protein. Accordingly, the expression of Cos2(900–1201), which binds Smo but not Ci, did not repress constitutiveCi activity. Furthermore, Cos2 (900–1201) did not allow Hh signal-dependent activation (Fig. 4c), indicating competition with endoge-nous Cos2 protein for Smo-binding sites. Fig. 4d confirmed that bothCos2 (900–1201) and Cos2 (1–1201), but not Cos2 (1–900), competewith endogenous Cos2 protein binding on Smo. Our data indicate thatthe association of Cos2 with Smo is necessary for the Hh-dependentregulation of Ci and confirm that Hh signalling regulates associationbetween all four proteins.

Smo stabilization at the plasma membrane seems to be a crucial stepin activation of the Hh pathway11. Hh-regulated vesicle transport ofSmo to the plasma membrane has been suggested as a possible mecha-nism for the accumulation and stabilization of Smo10,11. Our dataindicate that Smo is directly associated with the Cos2–Fu–Ci complex,both in the vesicular cytoplasmic pool and at the plasma membrane.So, how is the Hh signal transmitted from Smo to Ci? An attractivemodel suggests that, after Hh activation, an intracellular complexcomprising all four proteins — possibly associated with vesicles —might be shuttled to the plasma membrane as a result of increasedtransport towards and/or decreased recycling away from the plasmamembrane. The Cos2–Fu–Ci complex might also shuttle independ-ently of Smo, because RNAi to Smo does not change the distribution ofthe Cos2–Fu complex (data not shown). The stabilization of Smo atthe plasma membrane favoured the destabilization of Cos2 and Fu thatoccurs at the plasma membrane or during the transport from cytosol.Inhibition of Cos2 in the complex would allow the dissociation of Ci,leading to its activation and translocation to the nucleus. The differentsubcellular localization of the complex when cells are not activated byHh might allow the regulation of Ci by protein kinase A andSuppressor of fused28. In this model Cos2 could have a central role as ascaffold protein that brings together negative and positive regulators ofthe pathway.

METHODSGenetics. All alleles used were null: ptcIIW , cosW1 and smoIIX43. Transgenes wereas follows: UAS hh MI, UAS smo (ref. 18), UAS cos and UAS rab7. To generatemutant clones, larvae 24–36 h old with the following genotype were heatshocked at 37 °C for 1 h: y, hs-Flp122; FRT42D arm LacZ/FRT42D ptcIIW, y w hs-Flp122; NM(myc)31 EPw+ FRT40A/smoIIX43 FRT 40A, y, hs-Flp122; FRT42D armLacZ/FRT42D cosW1. Overexpressing clones were generated by means of the‘FLP-out’ technique29 with the use of the following lines: y; act<CD2<GAL4,UASGFPnls and y w hs-Flp122; Sp/SM6-TM6B. All clones were generated on

larvae 24–36 h old with 1 h of heat shock at 35 °C.

Immunolabelling. Embryos were immunostained as described in ref. 30.Antibodies were used at the following dilutions: mouse monoclonal JB10 anti-Nrt, 1:400 (gift from A. LeBivic); rabbit polyclonal anti-Hh, 1:200 (ref. 30); ratanti-Smo, 1:500 (ref. 10); purified rabbit anti-Smo, 1:200 (home-made); puri-fied rabbit anti-Cos, 1:200 (home-made); purified rabbit anti-Fu, 1:250 (home-made); monoclonal 2A1 rat anti-Ci, 1:20 (gift from R. Holmgren); monoclonal4D9 mouse anti-En, 1:20 (obtained from Developmental Studies HybridomaBank); monoclonal 5E10 mouse anti-Ptc, 1:200 (ref. 31); monoclonal mouseanti-Myc, 1:200 (Santa Cruz); monoclonal mouse anti-β-Gal, 1:1,000(Promega); rabbit anti-β-Gal 1:1,000 (Capel). Secondary antibodies coupledwith fluorescent Cy5 and Cy3 (Jackson) or alexa488 (Molecular Probes) wereused at a dilution of 1:200. Images were obtained by confocal microscopy (LeicaDMR TCS_NT). Subapical slides in Fig. 3 were observed with a 100× objectiveand a numeric zoom of 2. Images in Fig. 3e, h were obtained with a PhotoShopsoftware treatment that kept only the Smo–Cos2 or Ci–Cos2 overlaps. Thesame results were obtained with the Metamorph system.

Production of antisera. Rabbit antisera to Smo, Cos2 and Fu were raised againstpolyhistidine fusion proteins. His–Cos2 (residues 509–751), His–Fu (residues420–583) and His–Smo (residues 738–1031) were constructed into pET-30b(Novagen). Fusion proteins were produced in Escherichia coli strain BL21(DE3)before immunization. Antibodies were affinity purified with standard procedures.

Cell culture and transfections. Drosophila S2 and cl-8 cells were maintained asdescribed in ref. 32. Hh-N-conditioned media were prepared as published21

and used for Figs 2b and 4c. For other experiments (Figs 2a, 3a and 3b) a stablecl-8 hh-expressing cell line has been established. The 1.6-kilobase MseI frag-ment of hh cDNA in pBluescript SK+ (Stratagene) corresponding to the entirecoding region was cloned into the EcoRV sites of pMT/V5–His (Invitrogen).This vector is designed to express proteins under control of the metallothioneinpromoter. Stable transformed cl-8 cell lines were selected with hygromycin32.Transformed cells were examined for Hh expression after metal-inducible geneexpression (with 0.1 mM cadmium chloride). For transient expression,smo–haemagglutinin (HA) and cos2–myc epitope-tagged cDNAs were sub-cloned into the Drosophila expression vectors pAct–Myc or pAct–HA designedto express proteins under the control of the actin constitutive promoter. Full-length cos2 and fragments thereof (amplified by polymerase chain reaction(PCR)) were subcloned into pAct–Myc in frame with the Myc epitope. For thecompetition assay smo–HA and cos2–myc constructs were transfected at a ratioof 1:10.

RNA interference in S2 cells and luciferase assay. Double-stranded RNAs wereproduced by transcription in vitro with T7 polymerase on PCR products corre-sponding to amino acids 621–841 of Cos2, amino acids 740–970 of Ptc andamino acids 141–370 of Smo. Transfection with dsRNAi into S2 cells were asdescribed previously24. After 1 day of incubation with dsRNAi, 2 µg of 8 × Gliluciferase reporter23, 0.1 µg of ci expression vector (pAct ci) and 2 µg of pActlacZ (to normalize for transfection efficiency) were transfected and incubatedfor 24 h. Transfected cells were then split into control or Hh-N-containingmedia and incubated for an additional 16 h. Samples were prepared for analysisby SDS–polyacrylamide-gel electrophoresis, and luciferase activity was meas-ured with Luciferase Assay Kits (Promega).

Lysis, immunoprecipitation and fractionation of cellular extracts. Samples inFig. 2a were prepared in RIPA buffer. For immunoprecipitation analysis, cellextracts were made with a lysis buffer consisting of 20 mM Hepes pH 7.5,150 mM NaCl, 1 mM EGTA and 1% Triton X-100. Samples were quantified andanalysed by western blot performed as described previously21 with polyclonalrabbit anti-Cos2 (dilution 1:5,000), rabbit anti-Fu (1:1,000), monoclonal 2A1rat anti-Ci (1:20), goat anti-Smo (1:500; Santa Cruz), mouse anti-Myc (1:1,000)and mouse anti-HA (1:1,000) antibodies. For immunoprecipitations, 10 µl ofprotein G–Sepharose bound to antibodies were added to the clarified cell lysatesat 4 °C for 2 h and immuno-complexes were washed five times with lysis buffer.Enhanced chemiluminescence reagents were used for antibody detection afterblotting to nitrocellulose membranes. Subcellular fractions were prepared asdescribed previously25.

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L E T T E R S

NATURE CELL BIOLOGY VOLUME 5 | NUMBER 10 | OCTOBER 2003 913

Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGEMENTSWe thank A. Le Bivic and S. Cohen for antibodies; M. van den Heuvel, K. Ho, M.Scott, K. Basler, P. Ingham, M. Gonzalez-Gaitan and I. Guerrero for fly stocks; all‘fly’ members of the ISDBCR for exciting discussions about this work; andP. Léopold, J. P. Vincent, S. Eaton, A. Goldsborough and N. Tapon for comments onthe manuscript. R.R. is supported by a doctoral fellowship from the FrenchResearch Ministry. This work was supported by grants from the ‘Association pourla Recherche sur le Cancer’, ‘la Fondation de France’ and the ATIPE programme ofthe Centre National de la Recherche Scientifique to P.P.T.

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

Received 1 August 2003; accepted 21 August 2003Published online at http://www.nature.com/naturecellbiology

1. Ingham, P. W. & McMahon, A. P. Hedgehog signaling in animal development: para-digms and principles. Genes Dev. 15, 3059–3087 (2001).

2. Tabata, T., Eaton, S. & Kornberg, T. B. The Drosophila hedgehog gene is expressedspecifically in posterior compartment cells and is a target of engrailed regulation.Genes Dev. 6, 2635–2645 (1992).

3. Lee, J. J., von Kessler, D. P., Parks, S. & Beachy, P. A. Secretion and localized tran-scription suggest a role in positional signaling for products of the segmentation genehedgehog. Cell 71, 33–50 (1992).

4. Dominguez, M., Brunner, M., Hafen, E. & Basler, K. Sending and receiving the hedge-hog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272,1621–1625 (1996).

5. Ingham, P. W., Taylor, A. M. & Nakano, Y. Role of the Drosophila patched gene in posi-tional signalling. Nature 353, 184–187 (1991).

6. Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M. & Tabin, C. J. Biochemical evi-dence that patched is the Hedgehog receptor. Nature 384, 176–179 (1996).

7. Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M. & Hooper, J. E. The Drosophilasmoothened gene encodes a seven-pass membrane protein, a putative receptor for thehedgehog signal. Cell 86, 221–232 (1996).

8. van den Heuvel, M. & Ingham, P. W. smoothened encodes a receptor-like serpentineprotein required for hedgehog signalling. Nature 382, 547–551 (1996).

9. Chen, Y. & Struhl, G. Dual roles for patched in sequestering and transducingHedgehog. Cell 87, 553–563 (1996).

10. Denef, N., Neubuser, D., Perez, L. & Cohen, S. M. Hedgehog induces oppositechanges in turnover and subcellular localization of patched and smoothened. Cell102, 521–531 (2000).

11. Zhu, A. J. et al. Altered localization of Drosophila Smoothened protein activatesHedgehog signal transduction. Genes Dev. 17, 1240–1252 (2003).

12. Robbins, D. J. et al. Hedgehog elicits signal transduction by means of a large complexcontaining the kinesin-related protein costal2. Cell 90, 225–234 (1997).

13. Sisson, J. C., Ho, K. S., Suyama, K. & Scott, M. P. Costal2, a novel kinesin-related

protein in the Hedgehog signaling pathway. Cell 90, 235–245 (1997).14. Wang, G., Amanai, K., Wang, B. & Jiang, J. Interactions with Costal2 and Suppressor

of fused regulate nuclear translocation and activity of Cubitus interruptus. Genes Dev.14, 2893–2905 (2000).

15. Alcedo, J., Zou, Y. & Noll, M. Posttranscriptional regulation of smoothened is part of aself-correcting mechanism in the Hedgehog signaling system. Mol. Cell 6, 457–465(2000).

16. Ingham, P. W. et al. Patched represses the Hedgehog signalling pathway by promotingmodification of the Smoothened protein. Curr. Biol. 10, 1315–1318 (2000).

17. Therond, P. et al. Molecular organisation and expression pattern of the segment polar-ity gene fused of Drosophila melanogaster. Mech. Dev. 44, 65–80 (1993).

18. Martin, V., Carrillo, G., Torroja, C. & Guerrero, I. The sterol-sensing domain of Patchedprotein seems to control Smoothened activity through Patched vesicular trafficking.Curr. Biol. 11, 601–607 (2001).

19. Strigini, M. & Cohen, S. M. A Hedgehog activity gradient contributes to AP axial pat-terning of the Drosophila wing. Development 124, 4697–4705 (1997).

20. Hooper, J. E. Smoothened translates Hedgehog levels into distinct responses.Development 130, 3951–3963 (2003).

21. Therond, P. P., Knight, J. D., Kornberg, T. B. & Bishop, J. M. Phosphorylation of thefused protein kinase in response to signaling from hedgehog. Proc. Natl Acad. Sci.USA 93, 4224–4228 (1996).

22. Chen, C. H. et al. Nuclear trafficking of Cubitus interruptus in the transcriptional reg-ulation of Hedgehog target gene expression. Cell 98, 305–316 (1999).

23. Sasaki, H., Hui, C., Nakafuku, M. & Kondoh, H. A binding site for Gli proteins isessential for HNF-3β floor plate enhancer activity in transgenics and can respond toShh in vitro. Development 124, 1313–1322 (1997).

24. Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell linesto dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503(2000).

25. Morfini, G., Szebenyi, G., Elluru, R., Ratner, N. & Brady, S. T. Glycogen synthasekinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-basedmotility. EMBO J. 21, 281–293 (2002).

26. Muller, H. A. & Wieschaus, E. armadillo, bazooka, and stardust are critical for earlystages in formation of the zonula adherens and maintenance of the polarized blasto-derm epithelium in Drosophila. J. Cell Biol. 134, 149–163 (1996).

27. Monnier, V., Ho, K. S., Sanial, M., Scott, M. P. & Plessis, A. Hedgehog signal trans-duction proteins: contacts of the Fused kinase and Ci transcription factor with thekinesin-related protein Costal2. BMC Dev. Biol. 2, 4–13 (2002).

28. Price, M. A., Kalderon, D. Proteolysis of the Hedgehog signaling effector Cubitusinterruptus requires phosphorylation by glycogen synthase kinase 3 and casein kinase1. Cell 108, 823–835 (2002).

29. Basler, K. & Struhl, G. Compartment boundaries and the control of Drosophila limbpattern by hedgehog protein. Nature 368, 208–214 (1994).

30. Gallet, A., Rodriguez, R., Ruel, L. & Therond, P. P. Cholesterol modification of hedge-hog is required for trafficking and movement, revealing an asymmetric cellularresponse to hedgehog. Dev. Cell 4, 191–204 (2003).

31. Strutt, H. et al. Mutations in the sterol-sensing domain of Patched suggest a role forvesicular trafficking in Smoothened regulation. Curr. Biol. 11, 608–613 (2001).

32. van Leeuwen, F., Samos, C. H. & Nusse, R. Biological activity of soluble wingless pro-tein in cultured Drosophila imaginal disc cells. Nature 368, 342–344 (1994).

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Figure S1. Destabilisation of Fu is depending upon Hh signaling. Allpictures are wing imaginal discs with posterior compartment on the right.(A-B) wild type discs showing stabilisation of Fu (in red) in most Acompartment but not in all cells at the A/P border labelled for Ptc in green.C-D are Z-sections of discs with apical position to the top (arrow marks thelimit of Fu stability). Fu is destabilised in Flp-out anterior clones of hh-

expressing cells (marked by GFP in E-F), in anterior clone of cellshomozygous mutant for ptc (G-H) and in Flp-out anterior clones of smo-expressing cells (I-J). Fu is stabilised in posterior clones of cells homozygousmutant for smo (K-L). Arrowheads indicate clones visualized by the presenceof GFP (E and I), and the absence of βgal (green in G), or Myc (green in K).Arrows (in A, B, E-L) mark the A/P border.