Hedgehog-induced phosphorylation by CK1 sustainsthe activity of Ci/Gli activatorQing Shia, Shuang Lia, Shuangxi Lia,b, Alice Jianga, Yongbin Chenc,d, and Jin Jianga,e,1

Departments of aDevelopmental Biology and ePharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235; bInstitute ofBiomedical Sciences, East China Normal University, Shanghai 200062, China; and cKey Laboratory of Animal Models and Human Disease Mechanisms anddYunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China

Edited by Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA, and approved November 11, 2014 (received for reviewAugust 28, 2014)

Hedgehog (Hh) signaling governs many developmental processesby regulating the balance between the repressor (CiR/GliR) andactivator (CiA/GliA) forms of Cubitus interruptus (Ci)/glioma-associated oncogene homolog (Gli) transcription factors. Althoughmuch is known about how CiR/GliR is controlled, the regulation ofCiA/GliA remains poorly understood. Here we demonstrate thatCasein kinase 1 (CK1) sustains Hh signaling downstream of Costal2and Suppressor of fused (Sufu) by protecting CiA from prematuredegradation. We show that Hh stimulates Ci phosphorylation byCK1 at multiple Ser/Thr-rich degrons to inhibit its recognition bythe Hh-induced MATH and BTB domain containing protein (HIB),a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases.In Hh-receiving cells, reduction of CK1 activity accelerated HIB-me-diated degradation of CiA, leading to premature loss of pathwayactivity. We also provide evidence that GliA is regulated by CK1 ina similar fashion and that CK1 acts downstream of Sufu to pro-mote Sonic hedgehog signaling. Taken together, our study notonly reveals an unanticipated and conserved mechanism by whichphosphorylation of Ci/Gli positively regulates Hh signaling butalso provides the first evidence, to our knowledge, that substraterecognition by the Cullin 3 family of E3 ubiquitin ligases is nega-tively regulated by a kinase.

Hedgehog | CK1 | Ci | Gli | SPOP

The evolutionarily conserved Hedgehog (Hh) signaling path-way governs embryogenesis and adult tissue homeostasis by

tightly controlling the balance between the repressor (CiR/GliR)and activator (CiA/GliA) forms of Cubitus interruptus (Ci)/Glitranscription factors (1–5). In Drosophila wing discs, Hh secretedfrom posterior (P) compartment cells moves into the anterior(A) compartment to form a local activity gradient near the A/Pboundary. Low, intermediate, and peak levels of Hh differen-tially regulate the CiR/CiA ratio to activate decapentaplegic (dpp),patched (ptc), and engrailed (en), respectively (6–8). In humans,imbalance between GliR and GliA causes various birth defectsand cancers (1, 9, 10).Generation of CiR/GliR occurs in the absence of Hh. The

kinesin-like proteins Costal2 (Cos2)/Kinesin superfamily member7 (Kif7) and the tumor suppressor Suppressor of fused (Sufu) formprotein complexes with full-length Ci/Gli (CiF/GliF) to prevent itsnuclear localization and promote its phosphorylation by multiplekinases, including Protein kinase A (PKA), Casein kinase 1 (CK1),and Glycogen synthase kinase 3 (GSK3), which targets it for Su-pernumerary limbs (Slimb)/β-Transducin repeat containing E3ubiquitin protein ligase (βTRCP)-mediated processing to generatetruncated repressor forms (11). The production of CiA/GliA

requires the binding of Hh ligand to the transmembrane receptorPtc, which alleviates the inhibition of the transmembrane signaltransducer Smoothened (Smo) by Ptc (1–3, 12, 13). Smoundergoes phosphorylation by multiple kinases that promoteits active conformation and cell surface (Drosophila)/primarycilium (vertebrates) accumulation (11, 14–20). Smo-mediatedintracellular signal transduction abrogates Ci/Gli processinginto CiR/GliR and converts accumulated full-length Ci/Gli into

CiA/GliA by dissociating Ci/Gli from Cos2/Kif7 and Sufu (8, 21–27). The Drosophila Ser/Thr kinase Fused (Fu) is required toantagonize Cos2- and Sufu-mediated inhibition of Ci (28–30),but its mammalian counterpart remains to be identified.CiA is unstable and is degraded by the ubiquitin/proteasome

pathway mediated by the MATH- and BTB-domain containingprotein HIB (also called “Rdx”) (8, 31, 32). Interestingly, HIB isup-regulated in response to Hh in both embryos and imaginaldiscs (31, 32), and HIB also down-regulates Sufu through Crn(33), thus forming feedback loops to fine-tune CiA activity.However, it is not clear how HIB-mediated degradation of CiA iskept in check to prevent premature loss of Hh signaling activity.CK1 plays a dual role in both Drosophila and vertebrate Hh

signaling (11). In the absence of Hh, CK1 phosphorylates Ci/Gliafter PKA-primed phosphorylation, which is essential for theproduction of CiR/GliR (34–38); however, in the presence of Hh,CK1 phosphorylates Smo and likely Fu, to activate the Hhpathway (15, 30, 39–41). Here we uncover an unanticipatedpositive role of CK1 in the regulation of CiA downstream of Smoand Fu. We show that reduction in CK1 activity leads to de-stabilization of CiA and diminished Hh pathway activity. Mecha-nistically, we provide biochemical evidence that CK1 phosphorylatesmultiple Ser/Thr-rich degrons in Ci to attenuate HIB recognitionand thus reduce the rate of HIB-mediated CiA degradation.Blockage of the HIB-mediated degradation either by inactivatingHIB or by mutating the HIB degrons bypasses the requirement ofCK1 in the stabilization of CiA. Importantly, we show that GliA isregulated by CK1 in a conserved manner and that CK1 positivelyregulates Gli activity in Sufu mutant cells.

Significance

Hedgehog (Hh) signaling controls development and tissue ho-meostasis through the Cubitus interruptus (Ci)/glioma-associatedoncogene homolog (Gli) transcription factors, and abnormal Gliactivity causes congenital diseases and cancers. Here we showthat Ci/Gli phosphorylation by Casein kinase 1 positively regu-lates Hh pathway activity, providing insights into the regulationof Ci/Gli activity. By showing that phosphorylation protects theCi/Gli activator from premature degradation, our study not onlysheds lights on how the production and degradation of Ci/Gliactivator are delicately balanced to achieve optimal pathwayactivity but also provides the first evidence (to our knowledge)that protein degradation by the Cullin 3 family of E3 ubiquitinligases is negatively regulated by phosphorylation.

Author contributions: Q.S. and J.J. designed research; Q.S., Shuang Li, Shuangxi Li, A.J.,and Y.C. performed research; Q.S., Shuang Li, Shuangxi Li, Y.C., and J.J. analyzed data;and Q.S. and J.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: jin.jiang@UTSouthwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416652111/-/DCSupplemental.

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ResultsCK1 and PKA Differentially Regulate Ci Levels in Hh-Receiving Cells. Inwild-type wing discs of late third-instar larvae, CiF is accumulatedin A-compartment cells near the A/P boundary because of theinhibition of Ci processing by Hh secreted from P-compartmentcells (arrows in Fig. 1 A and B) (7); however, in A-compartmentcells immediately adjacent to the A/P boundary that receive highlevels of Hh, Ci staining is diminished because of the conversion ofCiF into labile CiA (arrowheads in Fig. 1 A and B) (8), as evi-denced by the expression of the high-threshold Hh target gene enin these cells (arrowhead in Fig. 1B′).Inactivation of CK1 using a wing-specific Gal4 driver MS1096

to express CRL (MS > CRL), a UAS-CK1-RNAi transgene thatknocks down both CK1α and CK1e (35), ectopically stabilizedCiF in A-compartment cells distant from the A/P boundary(arrows in Fig. 1 C and D), consistent with previous findings thatphosphorylation of CiF by CK1 is required for its proteolyticprocessing (35). MS > CRL also reduced ptc expression at theA/P boundary and blocked Hh-dependent en expression in A-compartment cells (Fig. 1 C′ and D′), as is consistent with CK1having a positive role in Smo/Fu activation (30, 39–41). Similarresults were obtained when PKA was inactivated by expressinga mutant form of the PKA regulatory subunit (MS > R*) (Fig.S1) (39), because PKA also plays a dual role in Hh signaling by

regulating both Ci processing and Smo activation (34, 39, 42).Of note, CRL also affected Hh-independent en expressionin posterior (P) compartment cells as observed previously(Fig. 1D′) (39).The down-regulation of Hh target genes at the A/P boundary

of MS > CRL wing discs could be attributed to compromisedSmo/Fu activation and consequent failure of CiA production.However, we noticed that Ci levels in A-compartment cells nearthe A/P boundary were much lower than in A-compartment cellsaway from the boundary (arrowheads in Fig. 1 C and D), a resultthat would not be expected if MS > CRL blocked both Ci pro-cessing and CiF-to-CiA conversion. This finding is in sharp con-trast to the nearly uniform accumulation of Ci in A-compartmentcells of MS > R* wing discs (Fig. S1), a phenotype expected withthe blockage of both Ci processing and CiA production. Thus, themechanism(s) by which CRL diminishes Hh signaling activitymay differ from the mechanism(s) by which PKA is inactivated.Of note, coexpressing either CK1α or CK1e blocked CRL-medi-ated down-regulation of Ci in A-compartment cells near the A/Pboundary and rescued ptc and en expression (Fig. S1), confirmingthat the effect of CRL on Ci level and activity results from the lossof CK1α/e activity.That the destabilization of Ci by CRL occurs strictly in A-

compartment cells near the A/P boundary implies that this process

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Fig. 1. CK1 positively regulates Hh signaling at thelevel of Ci. (A–F′) Late third-instar wild-type (con-trol) wing discs or wing discs expressing the in-dicated transgenes under the control of MS1096Gal4 driver were immunostained to the show theexpression of Ci (red), Ptc (green), and En (blue). Inthis and following figures, wing discs are orientedwith anterior (A) to the left and posterior (P) to theright. The A compartment is marked by the ex-pression of Ci. In control wing discs, Ci is accumu-lated in A-compartment cells close to the A/Pboundary (arrows in A and B) but is down-regulatedin A-compartment cells abutting the A/P boundary(arrowheads in A and B). The arrowhead in B’ indi-cates en expression in A-compartment cells. CK1RNAi resulted in down-regulation of Ci in A-com-partment cells near the A/P boundary (arrowheadsin C and D). Fu RNAi or expression of SmoDN blockedthe down-regulation of Ci in A-compartmentcells near the A/P boundary caused by CK1 RNAi(arrowheads in E and F). CK1 RNAi blocked en ex-pression in A-compartment cells (D′). (G–J′) Latethird-instar cos22 mutant wing discs that expressedUAS-Sufu-RNAi (G–H′) or both UAS-Sufu-RNAi andCRL (I–J′) using MS1096 were immunostained toshow the expression of Ci (red), Ptc (green), and En(blue). Sufu RNAi in cos2 mutant discs induced ec-topic en expression but down-regulated the Ciprotein level (arrowheads in G, H, and H’). Com-bined RNAi of CK1 and Sufu in cos2 mutant discsblocked en expression in A-compartment cells (ar-rowhead in J′) and further reduced the Ci proteinlevel (arrowheads in I and J). Dashed lines demarcatethe A/P boundary (B, B′, D, and D′).

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is Hh-dependent. Therefore, it is possible that MS > CRL onlyattenuated but did not completely block Smo/Fu activation be-cause of incomplete CK1 knockdown and that the remaining Smo/Fu activity still could convert significant amounts of CiF into CiA,leading to reduced Ci levels at the A/P boundary. When CK1activity was compromised, CiA either was less active or was lostprematurely (see below), leading to diminished Hh pathway ac-tivity. Consistent with this notion, coexpression of Fu-RNAi ora dominant-negative form of Smo (SmoDN) with CRL abolishedHh-induced ptc expression but stabilized CiF at the A/P boundary(arrowheads in Fig. 1 E–F′), indicating that Smo and Fu were stillactivated, at least partially, to convert CiF into labile CiA in MS >CRL wing discs. Taken together, these results imply that CK1 mayhave additional positive role(s) in the Hh pathway downstreamof Smo/Fu.

CK1 Positively Regulates Ci Stability and Activity Downstream of Cos2and Sufu. To determine whether CK1 could exert a positive roledownstream of Smo/Fu, we first examined if CK1 is required foroptimal pathway activation elicited by constitutively active Smoand Fu. Expression of SmoSD (a Smo variant activated by con-verting PKA/CK1 phosphorylation clusters into acidic residues),SmoΔSAID (a Smo variant activated by deleting its auto-in-hibitory domain), or CC-FuEE (a Fu variant activated by forceddimerization in combination with phosphomimetic mutations inits kinase activation loop) using MS1096 induced ectopic ex-pression of all Hh target genes including ptc and en in A-com-partment cells (Fig. S2 A and A′, C and C′, and E and E′) (14, 23,39); however, the ectopic en expression was blocked by coex-pression of CRL (compare Fig. S2 B′, D′, F′with Fig. S2 A′, C′,and E′) (30), raising the possibility that CK1 is required to sus-tain Hh signaling downstream of these active forms of Smo/Fu.To test the possibility that CK1 positively regulates Hh sig-

naling downstream of Smo/Fu, we examined whether CK1 isrequired for Hh pathway activity in wing discs in which both Cos2and Sufu were inactivated so that Ci was constitutively activeindependent of Smo/Fu activation (1, 12). In cos2 mutant wingdiscs, Ci was accumulated uniformly in A-compartment cellsbecause Cos2 is required for Ci phosphorylation and proteolyticprocessing (22); ptc was ectopically expressed in A-compartmentcells at low levels because of accumulated CiF, but A-compart-ment en expression was lost because of compromised Fu acti-vation (29, 43). Expression of Sufu-RNAi using MS1096 in cos2mutant wing discs (referred to as “cos2 Sufu double-mutantdiscs”) resulted in increased expression of ptc (Fig. 1G′), ectopicexpression of en (arrowhead in Fig. 1H′), and concomitant re-duction of Ci staining in A-compartment cells (arrowheads inFig. 1 G and H) because of the conversion of CiF into labile CiA

in the absence of Sufu (8, 43). CRL blocked ectopic en expres-sion and reduced ptc expression in cos2 Sufu double-mutant discs(Fig. 1 I′ and J′). In addition, CRL further reduced endogenousCi levels in cos2 Sufu double-mutant discs (compare Fig. 1 I andJ with Fig. 1 G and H). Because conversion of CiF to CiA in thecos2 Sufu double-mutant background is independent of up-stream signaling, diminished Hh pathway activity and reduced Cistaining by CRL in this condition were likely caused by thepremature loss of CiA.

CK1 Regulates the Stability of CiA. To test further the possibilitythat CK1 protects CiA from premature loss, we expresseda processing-resistant form of Ci with three PKA sites (S838,S856, and S892) mutated to Ala (Ci-PKA) (42) in wing discs eitheralone or together with CRL using MS1096. Consistent with ourprevious findings, expression of Ci-PKA in P-compartment cellsactivated high levels of ptc expression (arrowhead in Fig. 2A′),suggesting that Ci-PKA was converted into CiA by Hh. Strikingly,CRL dramatically decreased the levels of Ci-PKA as well as ptcexpression in P-compartment cells (compare Fig. 2 B and B′ with

Fig. 2 A and A′), suggesting that CK1 is required for the main-tenance of activated Ci-PKA.Consistent with Fu being activated to generate labile CiA in

MS > CRL wing discs, coexpression of Fu-RNAi with CRL com-pletely blocked ptc expression but elevated Ci-PKA levels inP-compartment cells (Fig. 2 C and C′). The accumulation of Ci-PKA

in the P-compartment cells when both CK1 and Fu were inacti-vated suggests that CK1 activity is not required for the stabiliza-tion of inactive Ci-PKA. We found that expression of either CK1αor CK1e in CRL-expressing P-compartment cells rescued ptc ex-pression and restored Ci-PKA levels (Fig. S3), consistent with thenotion that both CK1 isoforms can promote CiA stabilization.

CK1 Protects CiA from HIB-Mediated Degradation. We have shownpreviously that CiA degradation is mediated by the Cullin 3(Cul3)-based E3 ubiquitination ligase that contains HIB (31). Todetermine whether the loss of CiA in CRL-expressing P-com-partment cells is caused by the accelerated degradation by HIB,we coexpressed HIB-RNAi and CRL with Ci-PKA. We found thatHIB inactivation restored Ci-PKA levels in CRL-expressing P-compartment cells (compare Fig. 2D with Fig. 2B). Unlike FuRNAi, which stabilized inactive Ci-PKA (Fig. 2 C and C′), HIBRNAi stabilized Ci-PKA in an active form, as indicated by theectopic expression of ptc (Fig. 2D′). An HIB-binding–deficientform of Ci (Cim1-6) (44) remained stable and induced ectopicptc expression in CRL-expressing P-compartment cells (Fig. 2 E–F′). Furthermore, we found that combined knockdown of HIBand CK1 restored endogenous Ci and ptc expression in A-com-partment cells near the A/P boundary but failed to rescue CRL-suppressed en expression in A-compartment cells (Fig. S4), im-plying that CK1 may regulate Hh pathway activity positivelythrough an additional mechanism(s) that is independent of HIB.Taken together, these results suggest that CK1 is dispensable forCiA stability when HIB-mediated degradation is blocked.We next determined whether gain of CK1 function could

stabilize CiA. When expressed alone, Ci-PKA was down-regulatedin P-compartment cells because of its conversion into labile CiA

(Fig. S5). Indeed, inactivation of HIB by RNAi abolished thisdown-regulation, allowing Ci-PKA to accumulate at levels similar tothose in A-compartment cells (Fig. S5). Overexpression of eitherCK1α or CK1e also stabilized Ci-PKA in P-compartment cells (Fig.S5). Furthermore, overexpression of CK1 in the posterior regionof eye imaginal discs using the GMR Gal4 driver increased thelevels of endogenous Ci (Fig. S5), phenocopying HIB RNAi inthese cells (Fig. S5) (31) and suggesting that up-regulation of CK1activity could counteract HIB-mediated degradation of Ci.To test directly whether CK1 regulates the stability of acti-

vated Ci, we used a cell-based assay in which we measured thestability of Ci-PKA under Hh-stimulated and unstimulated con-ditions. S2 cells were transfected with HA-tagged Ci-PKA to-gether with Myc-CFP as an internal control. The cells weretreated with Hh-conditioned medium or control medium as wellas with CK1α/e dsRNA or luciferase (Luc) dsRNA as a control.Under these conditions, CK1 RNAi did not block Fu activationbecause Hh stimulated Fu phosphorylation in the presence ofCK1α/e dsRNA (Fig. S6). After cells were treated with cyclo-heximide (CHX) to block protein synthesis, HA-Ci-PKA proteinlevels were measured by Western blot at different time points.We found that Hh treatment accelerated the degradation of HA-Ci-PKA (compare Fig. 2I with Fig. 2G). HIB RNAi restored thestability of HA-Ci-PKA in the presence of Hh (compare Fig. 2Kwith Fig. 2I), consistent with the notion that Hh converts CiF intolabile CiA degraded by HIB. On the other hand, CK1 RNAifurther destabilized HA-Ci-PKA in the presence of Hh (compareFig. 2J with Fig. 2I) but did not affect the stability of HA-Ci-PKA

in the absence of Hh (Fig. 2H). Finally, inactivation of HIBrestored the stability of HA-Ci-PKA in the presence of both Hhand CK1α/e dsRNA (compare Fig. 2L with Fig. 2J). Knockdown

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efficiency for individual dsRNAs was confirmed by Western blotanalysis of epitope-tagged transgene expression (Fig. S7). Takentogether, these results strengthen the conclusion derived from invivo experiments that CK1 protects Hh-activated Ci by antago-nizing HIB-mediated degradation.

CK1 Regulates the Interaction Between Ci and HIB. HIB promotes Cidegradation by binding to both the N- and C-terminal regions of Cithrough multivalent interactions with its MATH domain (44). Todetermine whether CK1 attenuates HIB-mediated degradation byregulating HIB/Ci interaction, we carried out coimmunoprecipita-tion experiments to determine whether HIB/Ci interaction ismodulated by changes in CK1 activity. S2 cells were treated witha proteasome inhibitor, MG132, to stabilize the HIB/Ci complexbefore the immunoprecipitation assay. We found that the expres-

sion of Flag-CK1α, Flag-CK1e, or both reduced the amounts ofHA-HIB coimmunoprecipitated with Myc-Ci-PKA (Fig. 3A). Fur-thermore, expression of the kinase domain of Xenopus CK1e (re-ferred to as “CK1*”), which exhibits potent activity in vivo (45), alsoinhibited the interaction between Myc-Ci-PKA and a dimerized formof the HIB MATH domain (Flag-MATH-CC) (Fig. 3B) (44). Onthe other hand, CK1 RNAi enhanced the association between Myc-Ci-PKA and Flag-MATH-CC (Fig. 3C), suggesting that CK1 inhibitsHIB binding to Ci. Furthermore, CK1* inhibited HIB binding toboth the N-terminal and C-terminal regions of Ci (Fig. S8), sug-gesting that CK1 regulates HIB binding to multiple Ci domains.

CK1 Inhibits HIB Binding to Ci by Phosphorylating Multiple S/T-RichDegrons. Both the N- and C-terminal regions of Ci containa number of S/T-rich motifs that mediate HIB binding and Ci

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Fig. 2. CK1 protects CiA from HIB-mediated degra-dation. (A–F′) Late third-instar wing discs expressingthe indicated transgenes under the control ofMS1096were immunostained to show the expressionof Ci (red) and Ptc (green). Expression of Ci-PKA inP-compartment cells induced ectopic expression ofptc in these cells (arrowheads in A and A′). CK1 RNAidown-regulated the levels of Ci-PKA and abolishedthe ectopic expression of ptc in P-compartment cells(arrowheads in B and B′). Simultaneous knockdownof Fu and CK1 restored Ci-PKA level but not the ec-topic ptc expression (arrowheads in C and C′),whereas combined knockdown of HIB and CK1 re-stored both Ci-PKA and ptc expression (arrowheads inD and D′) in P-compartment cells expressing MS >Ci-PKA. Knockdown of CK1 did not significantly affectthe levels of Cim1-6 or ectopic ptc expression inducedby Cim1-6 in P-compartment cells (arrowheads inE–F′). (G–L) CK1 regulates the stability of Ci-PKA incells stimulated with Hh. Protein stability assaysfor Ci-PKA expressed in S2 cells. S2 cells treated withcontrol (luciferase) dsRNA (G and I), CK1α/e dsRNA(H and J), HIB dsRNA (K ), or CK1α/e + HIB dsRNAs(L) were transfected with HA-Ci-PKA and Myc-CFPexpression constructs. The transfected cells weretreated with control (G and H) or Hh-conditionedmedium (I–L). After treatment with CHX for theindicated periods of time, cell extracts were sub-ject to Western blot analysis with anti-HA andanti-Myc antibodies. Myc-CFP was used as an in-ternal control. Quantification of HA-Ci-PKA levelsat different time points is shown below each au-toradiogram. Data are means ± SD from threeindependent experiments.

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degradation, and these S/T-rich degrons also are present in otherHIB/SPOP substrates (44, 46). Interestingly, converting the S/Tresidues of HIB/SPOP degrons with either phosphorylated resi-dues or acidic residues to mimic phosphorylation blocked HIB/SPOP binding (44, 46), raising an interesting possibility thatsubstrate recognition by Cul3HIB/SPOP family of E3 ligases could

be inhibited by kinases that phosphorylate HIB/SPOP degrons.We noticed that many HIB/SPOP degrons in Ci contain CK1phosphorylation consensus sites: D/E/S(P)/T(P)[X1-3]S/T (bold-face letters represent CK1 phosphorylation sites) (Fig. 3F) (47),raising the possibility that CK1 may regulate HIB/Ci interaction bydirectly phosphorylating one or more HIB degrons.To test this hypothesis, we first examined whether CK1

phosphorylates CiA in vivo by monitoring the mobility shift ofHA-Ci-PKA in S2 cells in the absence or presence of CK1* usingthe phospho-tag gel that specifically retards phosphorylatedproteins (48). We found that coexpression of CK1* induceda mobility shift of HA-Ci-PKA but not HA-Cim1-6 (Fig. 3D),suggesting that CK1* stimulated Ci phosphorylation at HIBdegrons. We also found that Hh induced a mobility shift of HA-Ci-PKA, which was abolished by CK1 RNAi (Fig. 3E, comparelanes 3 and 4 with lanes 1 and 2), suggesting that Hh stimulatesCi-PKA phosphorylation through CK1. Mutating the HIB degrons(HA-Cim1-6) abolished the Hh-induced mobility shift (Fig. 3E,lanes 5–8), indicating that Hh stimulated Ci phosphorylation atHIB degrons. Taken together, these results suggest that CK1phosphorylates one or more HIB degrons in vivo, which isstimulated by Hh.We then determined which HIB degron was phosphorylated

by CK1 by applying an in vitro kinase assay in which GST-Cifusion proteins containing individual HIB degrons (S1–S6 inFig. 3 F and G) were incubated with a recombinant CK1 in thepresence of γ-32p-ATP. We found that all six HIB degrons can bephosphorylated by CK1 in vitro, with S2 and S4 exhibiting thestrongest and S6 exhibiting an intermediate level of phosphory-lation (Fig. 3G). We focused on S4 and S6 because our previousstudy indicated that they are strong HIB-binding sites and reg-ulate HIB-mediated Ci degradation in vivo (44). The putativeCK1 sites in S4 (S385, S387, and S388) and S6 (S1363, S1364, andS1365) were mutated to Ala to generate S4A and S6A. An in vitrokinase assay indicated that GST-S4A no longer was phosphory-lated by CK1, and GST-S6A exhibited greatly reduced phos-phorylation compared with GST-S6 (Fig. 3G).To determine whether CK1-mediated phosphorylation of S4/6

regulates HIB binding, we mutated CK1 sites in S4/6 to Asp (S4/6D)to mimic phosphorylation in the GST fusion proteins (GST-S4Dand GST-S6D) or in the context of HA-Ci-PKA (HA-Ci-PKAS4D6D).GST pull-down assays indicated that the phosphomimetic mutationsabolished HIB binding to the corresponding degrons (Fig. 3H).Furthermore, coimmunoprecipitation experiments revealed thatHA-Ci-PKAS4D6D pulled down less Flag-MATH-CC than HA-Ci-PKA

(Fig. 3I), consistent with the notion that phosphorylation of S4/6inhibits HIB/Ci interaction.

Phosphomimetic Ci Exhibits Delayed Degradation. To determinewhether CK1 protects Ci from HIB-mediated degradation byphosphorylating the S/T-rich degrons, we first compared the sta-bility of HA-Ci-PKAS4D6D with that of HA-Ci-PKA in S2 cells treatedwith Hh-conditioned medium. We found that HA-Ci-PKAS4D6D

exhibited increased stability compared with HA-Ci-PKA upon Hhstimulation (Fig. 4 A and B). Unlike HA-Ci-PKA, which was desta-bilized by CK1 RNAi, the stability of HA-Ci-PKAS4D6D was notsignificantly affected by CK1 RNAi (Fig. 4 A and B), suggesting thatphosphomimetic Ci-PKA is less dependent on CK1 for its durabilityin the presence of Hh.We next compared the stability of HA-Ci-PKAS4D6D with that

of HA-Ci-PKA in wing imaginal discs. To ensure similar levels oftransgene expression, we generated transformants for UAS-HA-Ci-PKA and UAS-HA-Ci-PKAS4D6D using the phiC31 integrationsystem in which the transgenes were inserted at the same genomelocus (49). We first expressed these transgenes in wing discs usinga weak Gal4 driver, C765 (17) and found that HA-Ci-PKAS4D6D

exhibited higher protein levels and induced higher levels of ectopicptc expression than HA-Ci-PKA in P-compartment cells (Fig. 4 C–D′).

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Fig. 3. CK1 promotes phosphorylation of Ci-PKA and inhibits recruitment ofHIB. (A and B) Overexpression of CK1 inhibited HIB/Ci association. S2 cellswere transfected with Myc-Ci-PKA with or without the indicated HIB and CK1expression constructs. After treatment with MG132 for 4 h, cell lysates weresubjected to immunoprecipitation and Western blot analysis using the in-dicated antibodies. Of note, both CK1α and CK1e are tagged by a Flag epi-tope, and CK1e overlaps with a nonspecific band (asterisk) detected by theanti-Flag antibody. (C) CK1 RNAi enhanced HIB/Ci association in response toHh stimulation. S2 cells were treated with the control (luciferase) or CK1 α/edsRNA before transfection with Myc-Ci-PKA and Flag-MATH-CC. The trans-fected cells were treated with or without Hh-conditioned medium, followedby immunoprecipitation and Western blot analysis using the indicatedantibodies. (D) CK1 induced phosphorylation of Ci-PKA but not Cim1-6. S2cells were cotransfected with the indicated constructs and were treated withMG132. Cell lysates were separated on Phos-tag–conjugated SDS/PAGE,followed by Western blot analysis with an anti-Myc antibody. (E) Hh stim-ulated phosphorylation of Myc-Ci-PKA but not Myc-Cim1-6 through CK1. S2cells treated with luciferase or CK1α/e dsRNA were cotransfected with theindicated Ci constructs and were treated with or without Hh-conditionedmedium. After treatment with MG132 for 4 h, cell lysates were prepared andseparated on Phos-tag–conjugated SDS/PAGE, followed by Western blotanalysis with an anti-Myc antibody. (F) A diagram of full-length Ci with sixHIB-binding sites (S1–S6) indicated by individual bars and the sequences ofindividual sites shown underneath. The S/T-rich sequences are underlined.(G) In vitro kinase assay using a recombinant CK1 and GST-Ci fusion proteinscontaining the indicated wild-type or mutated HIB-binding sites in thepresence of γ-[32P]ATP. (Left) Short (Top) or long (Middle) exposure of theautoradiograph is shown. (H) GST-Ci fusion proteins containing wild-type ormutated S4 or S6 were incubated with cell extracts derived from HIB-N–expressing S2 cells. Input and bound HIB-N proteins were analyzed by Westernblot using an anti-HA antibody. (I) S2 cells were transfected with Flag-MATH-CC alone or together with Myc-Ci-PKA or Myc-Ci-PKAS4D6D, followed by immu-noprecipitation and Western blot analysis using the indicated antibodies. Myc-Ci-PKAS4D6D pulled down less Flag-MATH-CC than Myc-Ci-PKA.

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Of note, expression of HA-Ci-PKAS4D6D also induced weak ec-topic ptc expression in A-compartment cells (Fig. 4D′), consis-tent with its being more stable than HA-Ci-PKA. It is likely thatoverexpressed Ci was partially converted into a labile active formin A-compartment cells away from the A/P boundary because ofthe limiting amount of endogenous Sufu.When expressed under the control of MS1096, both

HA-Ci-PKAS4D6D and HA-Ci-PKA accumulated at high levels andfully activated ptc expression in P-compartment cells (Fig. 4 E–F′).However, when CRL was coexpressed with HA-Ci-PKA, both Ciprotein level and ectopic ptc expression were diminished inP-compartment cells (Fig. 4 G and G′). In contrast, when CRLwas coexpressed with HA-Ci-PKAS4D6D, a significant amount ofHA-Ci-PKAS4D6D remained in P-compartment cells and inducedectopic ptc expression in these cells (Fig. 4 H and H′). However,phosphomimetic mutations at S4/6 did not render CiA com-pletely independent of CK1 in wing discs, because the levels ofHA-Ci-PKAS4D6D decreased in P-compartment cells expressingCRL (compare Fig. 4H with Fig. 4F). It is likely that CK1 canregulate CiA stability by phosphorylating other HIB-degronsthat are not altered in HA-Ci-PKAS4D6D. It is also possible thatCK1 phosphorylates additional target(s) to stabilize CiA.

CK1 Protects Gli2 from HIB/SPOP-Mediated Degradation. The task ofCi in Hh signaling is divided between two members of the Glifamily transcription factors in vertebrates, Gli2 and Gli3; Gli2contributes mainly to the activator form (GliA) and Gli3 to therepressor form (GliR) of Gli activities (1, 50). Both full-lengthGli2 and Gli3 are subjected to HIB/SPOP-mediated degradationin mammalian cultured cells as well as in Drosophila imaginaldiscs (26, 31, 51). The degradation of Gli2/3 by HIB/SPOP is alsomediated by multiple S/T-rich degrons resembling those in Ci

(44), raising the possibility that CK1 may play a conserved role inregulating Gli proteins. Consistent with this notion, we foundthat CK1α RNAi reduced Gli-luc reporter gene expressiondriven by a constitutively active form of Smo (SmoSD0-5) inwhich all the CK1/GRK2 phosphorylation sites were convertedto acidic residues (Fig. S9) (15), suggesting that CK1 has anadditional positive input in the Shh pathway downstream of Smo.To test the possibility that CK1 controls the stability of GliA,

we took advantage of the observations that Gli proteins werecontrolled by Hh pathway components when expressed in Dro-sophila (31, 52). Consistent with a previous finding (52), ex-pression of Myc-tagged Gli2 in wing discs (MS > Myc-Gli2)induced ectopic ptc expression in P-compartment cells (Fig. 5A–B′′). Coexpression of CRL diminished Myc-Gli2 levels andblocked Gli2-induced ectopic ptc expression in these cells (Fig. 5C–D′′), suggesting that CK1 inactivation resulted in the loss ofactive Gli2. Coexpression of HIB-RNAi with CRL prevented Gli2degradation and restored the ectopic expression of ptc inP-compartment cells (Fig. 5 E–F′′), suggesting that CK1 protectsactive Gli2 from HIB-mediated degradation.We then asked whether CK1 attenuates HIB/SPOP-mediated

degradation of Gli2 by inhibiting its binding to Gli2 (44).Coexpression of Flag-SPOP with Myc-Gli2 in S2 cells diminishedMyc-Gli2 protein levels (Fig. 5G) (44), which can be partiallyrestored by coexpression of CK1* (Fig. 5G), suggesting that CK1attenuates SPOP-mediated degradation of Gli2. In S2 cellstreated with MG132, expression of CK1* reduced the amountsof Myc-Gli2 bound to Flag-SPOP or Flag-MATH-CC (Fig. 5 Hand I), suggesting that CK1 inhibits the recognition of Gli2 bySPOP. Similar results were obtained with Gli3 (Fig. S10).

A B

C C’ E E’ G G’

D D’ F F’ H H’

Fig. 4. Phosphomimetic Ci exhibits increased stability. (A) Protein stability assays for Ci-PKA and Ci-PKAS4D6D. S2 cells treated with or without CK1α/e dsRNAwere transfected with expression constructs for HA-Ci-PKA or HA-Ci-PKAS4D6D and Myc-CFP (as an internal control). After treatment with Hh-conditionedmedium for 24 h, the transfected cells were treated with CHX for the indicated time periods, followed by Western blot analysis with anti-HA and anti-Mycantibodies. The loading was normalized by Myc-CFP. (B) Quantification of HA-Ci-PKA and HA-Ci-PKAS4D6D levels at different time points. Data are means ± SDfrom three independent experiments. (C–H′) Late third-instar wing discs expressing HA-Ci-PKA (C and C′, E and E′, and G and G′) or HA-Ci-PKAS4D6D (D and D′, Fand F′, and H and H′) under the control of C765 (C–D′) or MS1096 (E–H′) in the absence (C–F′) or presence (G–H′) of CRL were immunostained to show theexpression of Ci (C–H) and Ptc (C′–H′). When expressed at lower levels, Ci-PKAS4D6D exhibited increased abundance and activated higher levels of ptc than Ci-PKA

(compare D–D′ with C–C′). Ci-PKAS4D6D also is more stable than Ci-PKA in P-compartment cells expressing CRL (compare H–H′ with G–G′).

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CK1 Acts Downstream of Sufu to Regulate GliA Activity. In Sufumutant cells, GliA is constitutively produced independent ofupstream signaling components (26, 53–55); therefore, we firsttested whether CK1 regulates endogenous GliA activity in Sufu−/−

mouse embryonic fibroblasts (MEFs) using a Gli-luc reporter as-say. Consistent with previous findings (26, 53), Sufu−/− MEFsexhibited high basal Gli-luc activity that was suppressed bytransfection with a mouse Sufu (mSufu) expression construct(Fig. 5J). To inactivate CK1, we transfected Sufu−/− MEFs withdominant-negative forms of CK1α (DN-CK1α) and CK1δ (DN-CK1δ) either individually or in combination. A previous studyshowed that DN-CK1α and DN-CK1δ selectively inhibit CK1αand CK1δ/e, respectively (56). As shown in Fig. 5J, DN-CK1αslightly reduced but DN-CK1δ did not significantly alter Gli-luc

activity in Sufu−/− MEFs; however, their combined expressionresulted in significant reduction of the Gli-luc activity, suggestingthat both CK1α and CK1δ/e are involved in preserving GliA

activity in Sufu−/− MEFs. Consistent with notion, overexpressionof CK1α in Sufu−/− MEFs increased Gli-luc activity (Fig. 5J).We also tested whether CK1 regulates GliA derived from

exogenously expressed Gli2. Transfecting Sufu−/− MEFs witha Gli2 expression construct greatly increased Gli-luc activity (Fig.5K). Coexpression of DN-CK1α and DN-CK1δ in combinationsuppressed but coexpression of wild-type CK1α increased Gli-lucactivity induced by Gli2, suggesting that CK1 promotes Gli2A

activity downstream of Sufu. Importantly, Gli-luc activity in-duced by Gli25m, a Gli2 variant containing substitutions in fiveS/T-rich degrons and resistant to SPOP-mediated degradation

A A’ A’’

C

E

C’ C’’

G H I

J K L

E’ E’’

B B’ B’’

D D’ D’’

F F’ F’’

Fig. 5. CK1 prevents HIB/SPOP-mediated down-regulation of Gli2 and promotes Hh signaling downstream of Sufu. (A–F′′) Late third-instar wing discsexpressingMS >Myc-Gli2 (A–B′′),MS >Myc-Gli2 + CRL (C–D′′), orMS >Myc-Gli2 + CRL + HIB-RNAi (E–F′′) were immunostained to show the expression of Myc-Gli2 (green), Ci (red), and Ptc (blue). Arrowheads indicate P-compartments marked by the lack of Ci expression. Myc-Gli2 levels and Gli2-induced ectopic ptcexpression in P-compartment cells were down-regulated by inactivation of CK1 (compare C–D′′with A–B′′). Simultaneous inactivation of HIB and CK1 restoredMyc-Gli2 protein levels and ectopic ptc expression in P-compartment cells (E–F′′). (G) CK1 inhibits SPOP-mediated down-regulation of Gli2. S2 cells weretransfected with Myc-Gli2 and Myc-CFP (as an internal control) with or without Flag-SPOP and CK1*. Coexpression of SPOP selectively down-regulated Myc-Gli2 but not Myc-CFP, and this down-regulation was attenuated by CK1* coexpression. (H and I) CK1 inhibits Gli2/SPOP association. S2 cells were transfectedwith Myc-Gli2 and Flag-SPOP (H) or Flag-MATH-CC (I) in the absence or presence of CK1*. Cell lysates were immunoprecipitated and blotted with the in-dicated antibodies. (J) Gli-luciferase (Gli-luc) reporter assay in Sufu−/− MEFs transfected with the indicated constructs. Gli luciferase activities were normalizedto Renilla luciferase activities. Combined expression of DN-CK1α and DN-CK1δ inhibited but overexpression of Flag-CK1α increased Gli-luc reporter geneexpression. (K and L) Gli-luc reporter assay in Sufu−/− MEFs transfected with Myc-Gli2 (K) or Myc-Gli25M (L), in the absence or presence of DN-CK1α and/or DN-CK1δ or Flag-CK1α coexpression. The activity of Myc-Gli2 but not of Myc-Gli25m was influenced by changing CK1 activity. Data are means ± SD from threeindependent experiments.

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(44), was not significantly affected by either gain or loss ofCK1 activity (Fig. 5L), further strengthening the notion thatCK1 promotes GliA activity by attenuating SPOP-mediateddegradation.

DiscussionThe Ci/Gli family of transcription factors regulates animal de-velopment through the canonical Hh signaling pathway, andtheir activities are tightly controlled by different levels of Hhmorphogen to elucidate distinct developmental outcomes. It hasbeen well established that phosphorylation-mediated proteolysisof Ci/Gli plays an inhibitory role in Hh signaling by keepingthe basal Hh pathway activity in check (11). Here we uncovered apreviously unidentified function of phosphorylation in the reg-ulation of Ci/Gli activator activity, i.e., the protection of CiA/GliA

from premature degradation. We provide evidence that Hhstimulates CK1-mediated phosphorylation of Ci, likely at mul-tiple S/T-rich degrons, and that these phosphorylation eventsattenuate the recruitment of HIB/SPOP, thus slowing theCul3HIB/SPOP-mediated degradation of CiA (Fig. 6). We proposethat CK1-mediated phosphorylation of CiA increases its stability,allowing CiA levels to exceed critical thresholds to activate Hhtarget genes. We also provide evidence that CK1 plays a con-served role in the regulation of GliA.CK1 was identified initially as a negative regulator of the Hh

signaling pathway that phosphorylates Ci at multiple sites fol-lowing the primed phosphorylation by PKA (35, 57). The se-quential phosphorylation of Ci by PKA, CK1, and GSK3 recruitsSCFSlimb/βTRCP that targets Ci for ubiquitin/proteasome-medi-ated processing to generate CiR (Fig. 6A) (35, 58, 59). Later,several studies uncovered positive roles of CK1 in Hh signalingin which CK1 phosphorylates and activates Smo and possibly Fu(14, 30, 39–41). Therefore, it was unexpected that inactivation ofCK1 compromised the Hh pathway activity elicited by constitu-tively activated forms of Smo and Fu or by simultaneous in-activation of Cos2 and Sufu. To uncouple the positive role ofCK1 in Hh signaling from its role in the regulation of Ci pro-cessing, we examined the consequences of CK1 inactivation onHh signaling in P-compartment cells that do not express endog-enous Ci but instead express an unprocessed form of Ci (Ci-PKA)from a transgene. We found that inactivation of CK1 blockedCi-PKA-induced ectopic ptc expression in P-compartment cells. It isunlikely that the loss of ectopic ptc expression caused by CK1RNAi results from the blockage of conversion of CiF into CiA

because one would expect elevated levels of Ci-PKA if such werethe case. Instead, we observed diminished levels of Ci-PKA whenCK1 was inactivated by CRL. Strikingly, coexpression of Fu-RNAiwith CRL restored Ci-PKA protein level but not ectopic ptc ex-pression. We interpret these results as showing that Ci-PKA wasstill converted into CiA in P-compartment cells expressing CRL,likely because residual CK1 activity sufficed to activate Smo andFu; however, CiA was degraded more rapidly when CK1 activitywas compromised, leading to a premature loss of Hh pathwayactivity (Fig. 6B). In P-compartment cells coexpressing Fu-RNAiand CRL, Ci-PKA no longer was converted into CiA because of thecomplete loss of Fu activity and was accumulated in an inactiveform that did not rely on CK1 for its stability. Hence, CK1 isspecifically required for the stabilization of CiA. This notion wasconfirmed by the cell-based assay in which we directly measuredwhether inactivation of CK1 altered the stability of Ci-PKA. Ourresults clearly showed that inactivation of CK1 shortened the half-life of Ci-PKA only in the presence of Hh signaling activity, sug-gesting that CK1 activity is required to extend the lifetime of Hh-activated Ci. We found that CK1 RNAi reduced Ci staining in A-compartment cells in which both Cos2 and Sufu were inactivated,suggesting that CK1 is required for the stabilization of CiA derivedfrom endogenous Ci.

Previous studies suggested that CiA degradation is mediatedby the Cul3-based E3 ubiquitin ligase Cul3HIB/SPOP and thatCul3/HIB-mediated degradation serves as a mechanism for ter-minating Hh pathway activity, which is essential for normalDrosophila eye development (31, 32, 60, 61). HIB expression isup-regulated by Hh signaling in embryos as well as in imaginaldiscs; thus, Cul3/HIB forms a negative feedback loop to fine-tune Hh pathway activity in both embryonic and imaginal diskdevelopment (31, 32). SPOP also is involved in the degradationof active forms of Gli proteins, because removal of SPOP in Sufumutant cells stabilized full-length Gli, leading to elevated Hhpathway activity (26). Aside from the observation that HIB is up-regulated by Hh, it is not clear whether Cul3HIB/SPOP-mediateddegradation of Ci/Gli is regulated by other mechanisms duringdevelopment. Here, we demonstrate that CK1 counteractsCul3HIB/SPOP-mediated degradation of CiA/GliA to prevent pre-mature loss of Hh signaling activity. Mechanistically, we showedthat CK1 attenuated binding of HIB/SPOP to Ci/Gli, likely byphosphorylating multiple S/T-rich degrons present in Ci/Gli2,although it remains possible that CK1 has additional target sites.Interestingly, we found that Hh induced phosphorylation ofCi-PKA, which was abolished by CK1 RNAi. These phosphory-lation events are distinct from previously characterized PKA-primed CK1 phosphorylation of Ci and appear to occur atmultiple S/T-rich degrons. Hence, Ci possesses two sets of CK1sites that play opposing roles in the Hh pathway and are regu-lated by Hh signaling in the opposite directions: (i) PKA-primedCK1 phosphorylation negatively regulates Ci activity by targetingit for Slimb-mediated processing to generate CiR, and thesephosphorylation events are inhibited by Hh; (ii) CK1-mediatedphosphorylation of Ci at multiple S/T-rich degrons preserves CiA

activity by attenuating HIB/SPOP-mediated degradation, andthese phosphorylation events are stimulated by Hh (Fig. 6A). Ofnote, removing HIB in MS > CRL wing discs failed to rescue enexpression in A-compartment cells (Fig. S4), which requires thehighest levels of Hh pathway activity. One possible explanation is

A

B

Fig. 6. CK1 exerts both positive and negative roles in Hh signaling byphosphorylating multiple targets. (A) CK1 regulates Hh signaling at multiplelevels. In the absence of Hh, CK1 phosphorylates Ci/Gli to promote Slimb/βTRCP-mediated proteolytic processing that generates the repressor formsof Ci/Gli (step 1). In Hh-stimulated cells, CK1 phosphorylates Smo to promoteCi/Gli activation (step 2) and protects the activated Ci/Gli from HIB/SPOP-mediated degradation (step 3). (B, Left) Under normal circumstances, CK1attenuates HIB/SPOP-mediated degradation of Ci/Gli, allowing CiA/GliA toaccumulate above certain thresholds necessary for the expression of Hhtarget genes. (Right) When CK1 activity is reduced, CiA/GliA no longer isprotected, leading to accelerated degradation of CiA/GliA by HIB/SPOP andpremature loss of Hh pathway activity. See text for details.

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that upstream components such as Smo and Fu may not be fullyactivated in these rescue experiments. Another possibility is thatCK1 may positively regulate CiA activity through an additionalmechanism(s) independent of HIB. A recent study reported thatvertebrate Hh signaling conveys its gradient information byelaborately modulating multisite phosphorylation of Gli proteins(62). Thus, it would be interesting to determine if Hh-inducedCK1 phosphorylation of Ci directly contributes its optimaltranscriptional activity in addition to regulating its stability.How Hh does signaling differentially regulate these positive

and negative phosphorylation events? Previous studies revealedthat Ci and its kinases, including PKA, GSK3, and CK1, formprotein complexes scaffolded by Cos2 and that Hh signalinginduces either dissociation or composition change of thesecomplexes (22, 29, 63), thereby impeding PKA/GSK3/CK1-mediated Ci phosphorylation and proteolytic processing. It isthought that Ci/Gli forms a complex with Sufu in its inactive stateand that Hh activates Ci/Gli by dissociating it from Sufu, thusexposing CiA/GliA to HIB/SPOP and making it vulnerable forubiquitin/proteasome-mediated degradation (24, 29, 31). It ispossible that an Hh-induced change in the formation or com-position of Ci/Gli-Sufu complexes makes Ci/Gli more accessibleto CK1, allowing CK1-mediated phosphorylation to counteractHIB/SPOP and modulate the speed of Ci/Gli degradation (Fig. 6B).This delicate balance may ensure appropriate levels of CiA/GliA forHh signaling, and cells could change this balance to modulateHh responses. For example, in Drosophila eye imaginal discs,differentiating cells posterior to the morphogenetic furrow up-regulate HIB to dampen the response to Hh by degrading Ci(31, 32, 61). Our finding that the loss and gain of CK1 activitycan modulate the levels of CiA/GliA activity in opposite direc-tions raises an interesting possibility that altering CK1 activitymay serve as a mechanism for fine-tuning Hh responses incertain contexts.It has been well established that the Cul1-based E3 ubiquitin

ligase SCF complexes recognize substrates upon their phos-phorylation, thus linking protein phosphorylation to proteindegradation (64). Whether substrate recognition by otherCullin families of E3 ligases also is regulated by phosphory-lation remains largely unknown. A previous study showed thatreplacing the S/T residues in several SPOP degrons with phos-phorylated residues blocked binding to SPOP in vitro (46),raising an interesting possibility that recognition of HIB/SPOPsubstrates could be regulated by kinases. Here we provide thefirst evidence, to our knowledge, that substrate recognition byCul3-based E3 ligases is negatively regulated by a kinase. BecauseCul3HIB/SPOP regulates a large family of proteins, our study raisesan interesting possibility that the stability of other Cul3HIB/SPOP

targets might also be regulated by phosphorylation.

Materials and MethodsDrosophila Stocks and Transgenes. The following Drosophila stocks andtransgenes were used for this study: CRL, UAS-R*, UAS-SmoDN (Smo-PKA12),

and UAS-SmoSD (SmoSD123) (35, 39); UAS-Ci-PKA (42); UAS-SmoΔSAID (14);UAS-CC-FuEE (29); cos22 (65); UAS-HIB-RNAi (31); UAS-Cim1-6 and UAS-MATH-CC (44); UAS-CK1α, UAS-CK1«, and UAS-CK1*(UAS-XCK1«-KD) (45);UAS-Myc-Gli2 (52); UAS-Fu-RNAi (Vienna Drosophila Resource Center no.27663); and UAS-Sufu-RNAi (Bloomington Drosophila Stock Center no. 28559).Amino acid substitutions of multiple S/T-rich degrons were generated by PCR-based site-directed mutagenesis. UAS-HA-Ci-PKA and UAS-HA-Ci-PKAS4D6D wereinserted into the attP site at 71B as previously described (49).

Cell Culture, Luciferase Reporter Assay, Immunoprecipitation, Western Blot,and Immunostaining. Drosophila S2 cells were cultured in Drosophila SFM(Invitrogen) with 10% (vol/vol) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 24 °C. Transfection was carried out using the CalciumPhosphate Transfection Kit (Specialty Media) according to the manu-facturer’s instructions. Hh-conditioned medium was carried out as previouslydescribed (17). Sufu−/− MEFs transfection and the Gli-luciferase reporter as-say were carried out as described (66). Immunoprecipitation and Westernblot analysis were carried out using standard protocols as previously de-scribed (22). The Phos tag-conjugated SDS/PAGE analysis was performedaccording to standard protocols (48). Phos tag-conjugated acrylamide waspurchased from NARD Institute. Immunostaining of imaginal discs was car-ried out as described (67). Antibodies used for this study were mouse anti-Flag (M2; Sigma), rabbit anti-Flag (Thermo Scientific), mouse anti-HA (F7; SantaCruz), mouse anti-Myc (9E10; Santa Cruz), phospho-Fu (pT161/pT154) antibody(29), Rat anti-Ci, 2A1 (68), rabbit anti-CKIe (kindly provided by D. M. Virshup,Duke-NUS Graduate Medical School, Singapore), mouse anti-Ptc, and mouseanti-En (Developmental Studies Hybridoma Bank).

RNAi in Drosophila S2 Cells. DNA templates corresponding to the codingregions of CK1α (nucleotides 601–1014) and CK1e (nucleotides 834–1323)were generated by PCR and used to make dsRNA targeting the C terminus ofCK1α and CK1e, respectively. dsRNA targeting the Firefly Luciferase codingsequence was used as a control. dsRNAs were generated by MEGAscriptHigh-Yield Transcription Kit (AM1334; Ambion). For the RNAi knockdownexperiments, S2 cells first were cultured in serum-free medium containingdsRNA for 12 h at 24 °C. After FBS was added to a final concentration of10% (vol/vol), dsRNA-treated cells were cultured overnight before trans-fection with DNA constructs. After additional culturing for 2 d, cells werecollected for analysis.

In Vitro Kinase Assay, GST Pull Down, and Protein Stability Assay. For the invitro kinase assay, individual GST-fusion proteins bound to glutathione beadswere mixed with 0.1 mM ATP containing 10 mCi of γ-32p-ATP andrecombinant CK1 kinase (CK1δ; New England Biolabs) and were incubatedat 30 °C for 1.5 h in reaction buffer [20 mM Tris·HCl (pH 8.0), 2 mM EDTA,10 mMMgCl2, 1 mM DTT]. Reactions were stopped by adding 4× SDS loadingbuffer, and the mixture was boiled at 100 °C for 5 min. The phosphorylatedGST-fusion proteins were analyzed by autoradiography after SDS/PAGE. TheGST pull-down assay was carried out as described (44). The protein stabilityassay was carried out as described (18).

ACKNOWLEDGMENTS.We thank BingWang for assistance, Drs. R. Holmgren,J. Wu, and P. T. Chuang and the Developmental Studies Hybridoma Bankfor reagents, and the Vienna Drosophila Resource Center and BloomingtonDrosophila Stock Center for fly stocks. This work was supported by NationalInstitutes of Health Grants GM061269 and GM067045 and Welch Founda-tion Grant I-1603 (to J.J.), and National Science Foundation of China Grants31328017, 81322030, and 31271579 (to Y.C.).

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