Hedgehog-induced phosphorylation by CK1 sustains PNAS PLUS the activity of Ci/Gli activator

Qing 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 of Biomedical Sciences, East China Normal University, Shanghai 200062, China; and cKey Laboratory of Animal Models and Human Disease Mechanisms and dYunnan 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 review August 28, 2014) Hedgehog (Hh) signaling governs many developmental processes CiA/GliA by dissociating Ci/Gli from Cos2/Kif7 and Sufu (8, 21– by regulating the balance between the (CiR/GliR) and 27). The Drosophila Ser/Thr kinase Fused (Fu) is required to activator (CiA/GliA) forms of Cubitus interruptus (Ci)/glioma- antagonize Cos2- and Sufu-mediated inhibition of Ci (28–30), associated oncogene homolog (Gli) transcription factors. Although but its mammalian counterpart remains to be identified. much is known about how CiR/GliR is controlled, the regulation of CiA is unstable and is degraded by the ubiquitin/ A A Ci /Gli remains poorly understood. Here we demonstrate that pathway mediated by the MATH- and BTB-domain containing Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 protein HIB (also called “Rdx”) (8, 31, 32). Interestingly, HIB is A and Suppressor of fused (Sufu) by protecting Ci from premature up-regulated in response to Hh in both embryos and imaginal degradation. We show that Hh stimulates Ci phosphorylation by discs (31, 32), and HIB also down-regulates Sufu through Crn CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by (33), thus forming feedback loops to fine-tune CiA activity. the Hh-induced MATH and BTB domain containing protein (HIB), However, it is not clear how HIB-mediated degradation of CiA is a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases. kept in check to prevent premature loss of Hh signaling activity. In Hh-receiving cells, reduction of CK1 activity accelerated HIB-me- CK1 plays a dual role in both Drosophila and vertebrate Hh diated degradation of CiA, leading to premature loss of pathway signaling (11). In the absence of Hh, CK1 phosphorylates Ci/Gli activity. We also provide evidence that GliA is regulated by CK1 in after PKA-primed phosphorylation, which is essential for the a similar fashion and that CK1 acts downstream of Sufu to pro- R R – mote Sonic hedgehog signaling. Taken together, our study not production of Ci /Gli (34 38); however, in the presence of Hh, only reveals an unanticipated and conserved mechanism by which CK1 phosphorylates Smo and likely Fu, to activate the Hh – phosphorylation of Ci/Gli positively regulates Hh signaling but pathway (15, 30, 39 41). Here we uncover an unanticipated A also provides the first evidence, to our knowledge, that substrate positive role of CK1 in the regulation of Ci downstream of Smo recognition by the Cullin 3 family of E3 ubiquitin ligases is nega- and Fu. We show that reduction in CK1 activity leads to de- A tively regulated by a kinase. stabilization of Ci and diminished Hh pathway activity. Mecha- nistically, we provide biochemical evidence that CK1 phosphorylates Hedgehog | CK1 | Ci | Gli | SPOP multiple Ser/Thr-rich degrons in Ci to attenuate HIB recognition and thus reduce the rate of HIB-mediated CiA degradation. he evolutionarily conserved Hedgehog (Hh) signaling path- Blockage of the HIB-mediated degradation either by inactivating Tway governs embryogenesis and adult tissue homeostasis by HIB or by mutating the HIB degrons bypasses the requirement of A A tightly controlling the balance between the repressor (CiR/GliR) CK1 in the stabilization of Ci . Importantly, we show that Gli is and activator (CiA/GliA) forms of Cubitus interruptus (Ci)/Gli regulated by CK1 in a conserved manner and that CK1 positively transcription factors (1–5). In Drosophila wing discs, Hh secreted regulates Gli activity in Sufu mutant cells. from posterior (P) compartment cells moves into the anterior (A) compartment to form a local activity gradient near the A/P Significance boundary. Low, intermediate, and peak levels of Hh differen- R A tially regulate the Ci /Ci ratio to activate decapentaplegic (dpp), Hedgehog (Hh) signaling controls development and tissue ho- patched (ptc), and engrailed (en), respectively (6–8). In humans, R A meostasis through the Cubitus interruptus (Ci)/glioma-associated imbalance between Gli and Gli causes various birth defects oncogene homolog (Gli) transcription factors, and abnormal Gli and cancers (1, 9, 10). R R activity causes congenital diseases and cancers. Here we show BIOLOGY Generation of Ci /Gli occurs in the absence of Hh. The that Ci/Gli phosphorylation by Casein kinase 1 positively regu- DEVELOPMENTAL kinesin-like proteins Costal2 (Cos2)/Kinesin superfamily member lates Hh pathway activity, providing insights into the regulation 7 (Kif7) and the tumor suppressor Suppressor of fused (Sufu) form F F of Ci/Gli activity. By showing that phosphorylation protects the protein complexes with full-length Ci/Gli (Ci /Gli ) to prevent its Ci/Gli activator from premature degradation, our study not only nuclear localization and promote its phosphorylation by multiple sheds lights on how the production and degradation of Ci/Gli kinases, including Protein kinase A (PKA), Casein kinase 1 (CK1), activator are delicately balanced to achieve optimal pathway and Glycogen synthase kinase 3 (GSK3), which targets it for Su- activity but also provides the first evidence (to our knowledge) pernumerary limbs (Slimb)/β-Transducin repeat containing E3 β that protein degradation by the Cullin 3 family of E3 ubiquitin ubiquitin protein ligase ( TRCP)-mediated processing to generate ligases is negatively regulated by phosphorylation. truncated repressor forms (11). The production of CiA/GliA requires the binding of Hh ligand to the transmembrane receptor Author contributions: Q.S. and J.J. designed research; Q.S., Shuang Li, Shuangxi Li, A.J., Ptc, which alleviates the inhibition of the transmembrane signal and Y.C. performed research; Q.S., Shuang Li, Shuangxi Li, Y.C., and J.J. analyzed data; transducer Smoothened (Smo) by Ptc (1–3, 12, 13). Smo and Q.S. and J.J. wrote the paper. undergoes phosphorylation by multiple kinases that promote The authors declare no conflict of interest. its active conformation and cell surface (Drosophila)/primary This article is a PNAS Direct Submission. cilium (vertebrates) accumulation (11, 14–20). Smo-mediated 1To whom correspondence may be addressed. Email: [email protected]. intracellular signal transduction abrogates Ci/Gli processing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. R R into Ci /Gli and converts accumulated full-length Ci/Gli into 1073/pnas.1416652111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1416652111 PNAS | Published online December 15, 2014 | E5651–E5660 Downloaded by guest on October 1, 2021 Results regulating both Ci processing and Smo activation (34, 39, 42). CK1 and PKA Differentially Regulate Ci Levels in Hh-Receiving Cells. In Of note, CRL also affected Hh-independent en expression wild-type wing discs of late third-instar larvae, CiF is accumulated in posterior (P) compartment cells as observed previously in A-compartment cells near the A/P boundary because of the (Fig. 1D′) (39). inhibition of Ci processing by Hh secreted from P-compartment The down-regulation of Hh target genes at the A/P boundary cells (arrows in Fig. 1 A and B) (7); however, in A-compartment of MS > CRL wing discs could be attributed to compromised A cells immediately adjacent to the A/P boundary that receive high Smo/Fu activation and consequent failure of Ci production. levels of Hh, Ci staining is diminished because of the conversion of However, we noticed that Ci levels in A-compartment cells near CiF into labile CiA (arrowheads in Fig. 1 A and B)(8),asevi- the A/P boundary were much lower than in A-compartment cells denced by the expression of the high-threshold Hh target gene en away from the boundary (arrowheads in Fig. 1 C and D), a result in these cells (arrowhead in Fig. 1B′). that would not be expected if MS > CRL blocked both Ci pro- Inactivation of CK1 using a wing-specific Gal4 driver MS1096 cessing and CiF-to-CiA conversion. This finding is in sharp con- to express CRL (MS > CRL), a UAS-CK1-RNAi transgene that trast to the nearly uniform accumulation of Ci in A-compartment knocks down both CK1α and CK1e (35), ectopically stabilized cells of MS > R* wing discs (Fig. S1), a phenotype expected with CiF in A-compartment cells distant from the A/P boundary the blockage of both Ci processing and CiA production. Thus, the (arrows in Fig. 1 C and D), consistent with previous findings that mechanism(s) by which CRL diminishes Hh signaling activity phosphorylation of CiF by CK1 is required for its proteolytic may differ from the mechanism(s) by which PKA is inactivated. processing (35). MS > CRL also reduced ptc expression at the Of note, coexpressing either CK1α or CK1e blocked CRL-medi- A/P boundary and blocked Hh-dependent en expression in A- ated down-regulation of Ci in A-compartment cells near the A/P compartment cells (Fig. 1 C′ and D′), as is consistent with CK1 boundary and rescued ptc and en expression (Fig. S1), confirming having a positive role in Smo/Fu activation (30, 39–41). Similar that the effect of CRL on Ci level and activity results from the loss results were obtained when PKA was inactivated by expressing of CK1α/e activity. a mutant form of the PKA regulatory subunit (MS > R*)(Fig. That the destabilization of Ci by CRL occurs strictly in A- S1) (39), because PKA also plays a dual role in Hh signaling by compartment cells near the A/P boundary implies that this process

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CC’DD’ Fig. 1. CK1 positively regulates Hh signaling at the level 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 MS1096 Gal4 driver were immunostained to the show the expression of Ci (red), Ptc (green), and En (blue). In this and following figures, wing discs are oriented with anterior (A) to the left and posterior (P) to the EE’FF’ right. 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/P boundary (arrows in A and B) but is down-regulated in 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. CK1 RNAi resulted in down-regulation of Ci in A-com- partment cells near the A/P boundary (arrowheads GG’HH’ in C and D). Fu RNAi or expression of SmoDN blocked the down-regulation of Ci in A-compartment cells 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′) Late third-instar cos22 mutant wing discs that expressed UAS-Sufu-RNAi (G–H′) or both UAS-Sufu-RNAi and CRL (I–J′) using MS1096 were immunostained to II’JJ’ show 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 Ci protein level (arrowheads in G, H, and H’). Com- bined RNAi of CK1 and Sufu in cos2 mutant discs blocked en expression in A-compartment cells (ar- rowhead in J′) and further reduced the Ci protein level (arrowheads in I and J). Dashed lines demarcate the A/P boundary (B, B′, D,andD′).

E5652 | www.pnas.org/cgi/doi/10.1073/pnas.1416652111 Shi et al. Downloaded by guest on October 1, 2021 is Hh-dependent. Therefore, it is possible that MS > CRL only Fig. 2 A and A′), suggesting that CK1 is required for the main- PNAS PLUS attenuated but did not completely block Smo/Fu activation be- tenance of activated Ci-PKA. cause of incomplete CK1 knockdown and that the remaining Smo/ Consistent with Fu being activated to generate labile CiA in Fu activity still could convert significant amounts of CiF into CiA, MS > CRL wing discs, coexpression of Fu-RNAi with CRL com- leading to reduced Ci levels at the A/P boundary. When CK1 pletely blocked ptc expression but elevated Ci-PKA levels in activity was compromised, CiA either was less active or was lost P-compartment cells (Fig. 2 C and C′). The accumulation of Ci-PKA prematurely (see below), leading to diminished Hh pathway ac- in the P-compartment cells when both CK1 and Fu were inacti- tivity. Consistent with this notion, coexpression of Fu-RNAi or vated suggests that CK1 activity is not required for the stabiliza- a dominant-negative form of Smo (SmoDN)withCRL abolished tion of inactive Ci-PKA. We found that expression of either CK1α Hh-induced ptc expression but stabilized CiF at the A/P boundary or CK1e in CRL-expressing P-compartment cells rescued ptc ex- (arrowheads in Fig. 1 E–F′), indicating that Smo and Fu were still pression and restored Ci-PKA levels (Fig. S3), consistent with the activated, at least partially, to convert CiF into labile CiA in MS > notion that both CK1 isoforms can promote CiA stabilization. CRL wing discs. Taken together, these results imply that CK1 may have additional positive role(s) in the Hh pathway downstream CK1 Protects CiA from HIB-Mediated Degradation. We have shown of Smo/Fu. previously that CiA degradation is mediated by the Cullin 3 (Cul3)-based E3 ubiquitination ligase that contains HIB (31). To CK1 Positively Regulates Ci Stability and Activity Downstream of Cos2 determine whether the loss of CiA in CRL-expressing P-com- and Sufu. To determine whether CK1 could exert a positive role partment cells is caused by the accelerated degradation by HIB, downstream of Smo/Fu, we first examined if CK1 is required for we coexpressed HIB-RNAi and CRL with Ci-PKA. We found that optimal pathway activation elicited by constitutively active Smo HIB inactivation restored Ci-PKA levels in CRL-expressing P- and Fu. Expression of SmoSD (a Smo variant activated by con- compartment cells (compare Fig. 2D with Fig. 2B). Unlike Fu verting PKA/CK1 phosphorylation clusters into acidic residues), RNAi, which stabilized inactive Ci-PKA (Fig. 2 C and C′), HIB SmoΔSAID (a Smo variant activated by deleting its auto-in- RNAi stabilized Ci-PKA in an active form, as indicated by the hibitory domain), or CC-FuEE (a Fu variant activated by forced ectopic expression of ptc (Fig. 2D′). An HIB-binding–deficient dimerization in combination with phosphomimetic mutations in form of Ci (Cim1-6) (44) remained stable and induced ectopic its kinase activation loop) using MS1096 induced ectopic ex- ptc expression in CRL-expressing P-compartment cells (Fig. 2 E– pression of all Hh target genes including ptc and en in A-com- F′). Furthermore, we found that combined knockdown of HIB partment cells (Fig. S2 A and A′, C and C′, and E and E′) (14, 23, and CK1 restored endogenous Ci and ptc expression in A-com- 39); however, the ectopic en expression was blocked by coex- partment cells near the A/P boundary but failed to rescue CRL- pression of CRL (compare Fig. S2 B′, D′, F′with Fig. S2 A′, C′, suppressed en expression in A-compartment cells (Fig. S4), im- and E′) (30), raising the possibility that CK1 is required to sus- plying that CK1 may regulate Hh pathway activity positively tain Hh signaling downstream of these active forms of Smo/Fu. through an additional mechanism(s) that is independent of HIB. To test the possibility that CK1 positively regulates Hh sig- Taken together, these results suggest that CK1 is dispensable for naling downstream of Smo/Fu, we examined whether CK1 is CiA stability when HIB-mediated degradation is blocked. required for Hh pathway activity in wing discs in which both Cos2 We next determined whether gain of CK1 function could and Sufu were inactivated so that Ci was constitutively active stabilize CiA. When expressed alone, Ci-PKA was down-regulated independent of Smo/Fu activation (1, 12). In cos2 mutant wing in P-compartment cells because of its conversion into labile CiA discs, Ci was accumulated uniformly in A-compartment cells (Fig. S5). Indeed, inactivation of HIB by RNAi abolished this because Cos2 is required for Ci phosphorylation and proteolytic down-regulation, allowing Ci-PKA to accumulate at levels similar to processing (22); ptc was ectopically expressed in A-compartment those in A-compartment cells (Fig. S5). Overexpression of either cells at low levels because of accumulated CiF, but A-compart- CK1α or CK1e also stabilized Ci-PKA in P-compartment cells (Fig. ment en expression was lost because of compromised Fu acti- S5). Furthermore, overexpression of CK1 in the posterior region vation (29, 43). Expression of Sufu-RNAi using MS1096 in cos2 of eye imaginal discs using the GMR Gal4 driver increased the mutant wing discs (referred to as “cos2 Sufu double-mutant levels of endogenous Ci (Fig. S5), phenocopying HIB RNAi in discs”) resulted in increased expression of ptc (Fig. 1G′), ectopic these cells (Fig. S5) (31) and suggesting that up-regulation of CK1 expression of en (arrowhead in Fig. 1H′), and concomitant re- activity could counteract HIB-mediated degradation of Ci. duction of Ci staining in A-compartment cells (arrowheads in To test directly whether CK1 regulates the stability of acti- Fig. 1 G and H) because of the conversion of CiF into labile CiA vated Ci, we used a cell-based assay in which we measured the in the absence of Sufu (8, 43). CRL blocked ectopic en expres- stability of Ci-PKA under Hh-stimulated and unstimulated con- sion and reduced ptc expression in cos2 Sufu double-mutant discs ditions. S2 cells were transfected with HA-tagged Ci-PKA to- (Fig. 1 I′ and J′). In addition, CRL further reduced endogenous gether with -CFP as an internal control. The cells were BIOLOGY Ci levels in cos2 Sufu double-mutant discs (compare Fig. 1 I and treated with Hh-conditioned medium or control medium as well DEVELOPMENTAL J with Fig. 1 G and H). Because conversion of CiF to CiA in the as with CK1α/e dsRNA or luciferase (Luc) dsRNA as a control. cos2 Sufu double-mutant background is independent of up- Under these conditions, CK1 RNAi did not block Fu activation stream signaling, diminished Hh pathway activity and reduced Ci because Hh stimulated Fu phosphorylation in the presence of staining by CRL in this condition were likely caused by the CK1α/e dsRNA (Fig. S6). After cells were treated with cyclo- premature loss of CiA. heximide (CHX) to block protein synthesis, HA-Ci-PKA protein levels were measured by Western blot at different time points. CK1 Regulates the Stability of CiA. To test further the possibility We found that Hh treatment accelerated the degradation of HA- that CK1 protects CiA from premature loss, we expressed Ci-PKA (compare Fig. 2I with Fig. 2G). HIB RNAi restored the a processing-resistant form of Ci with three PKA sites (S838, stability of HA-Ci-PKA in the presence of Hh (compare Fig. 2K S856, and S892) mutated to Ala (Ci-PKA) (42) in wing discs either with Fig. 2I), consistent with the notion that Hh converts CiF into alone or together with CRL using MS1096. Consistent with our labile CiA degraded by HIB. On the other hand, CK1 RNAi previous findings, expression of Ci-PKA in P-compartment cells further destabilized HA-Ci-PKA in the presence of Hh (compare activated high levels of ptc expression (arrowhead in Fig. 2A′), Fig. 2J with Fig. 2I) but did not affect the stability of HA-Ci-PKA suggesting that Ci-PKA was converted into CiA by Hh. Strikingly, in the absence of Hh (Fig. 2H). Finally, inactivation of HIB CRL dramatically decreased the levels of Ci-PKA as well as ptc restored the stability of HA-Ci-PKA in the presence of both Hh expression in P-compartment cells (compare Fig. 2 B and B′ with and CK1α/e dsRNA (compare Fig. 2L with Fig. 2J). Knockdown

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Fig. 2. CK1 protects CiA from HIB-mediated degra- dation. (A–F′) Late third-instar wing discs expressing the indicated transgenes under the control of MS1096 were immunostained to show the expression of Ci (red) and Ptc (green). Expression of Ci-PKA in P-compartment cells induced ectopic expression of GHI ptc in these cells (arrowheads in A and A′). CK1 RNAi down-regulated the levels of Ci-PKA and abolished the ectopic expression of ptc in P-compartment cells (arrowheads in B and B′). Simultaneous knockdown of 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 in D and D′) in P-compartment cells expressing MS > Ci-PKA. Knockdown of CK1 did not significantly affect the levels of Cim1-6 or ectopic ptc expression induced by Cim1-6 in P-compartment cells (arrowheads in E–F′). (G–L) CK1 regulates the stability of Ci-PKA in cells stimulated with Hh. Protein stability assays for Ci-PKA expressed in S2 cells. S2 cells treated with JKLcontrol (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-CFP expression constructs. The transfected cells were treated with control (G and H) or Hh-conditioned medium (I–L). After treatment with CHX for the indicated periods of time, cell extracts were sub- ject to Western blot analysis with anti-HA and anti-Myc antibodies. Myc-CFP was used as an in- ternal control. Quantification of HA-Ci-PKA levels at different time points is shown below each au- toradiogram. Data are means ± SD from three independent experiments.

efficiency for individual dsRNAs was confirmed by Western blot sion of Flag-CK1α,Flag-CK1e, or both reduced the amounts of analysis of epitope-tagged transgene expression (Fig. S7). Taken HA-HIB coimmunoprecipitated with Myc-Ci-PKA (Fig. 3A). Fur- together, these results strengthen the conclusion derived from in thermore, expression of the kinase domain of Xenopus CK1e (re- vivo experiments that CK1 protects Hh-activated Ci by antago- ferred to as “CK1*”), which exhibits potent activity in vivo (45), also nizing HIB-mediated degradation. inhibitedtheinteractionbetweenMyc-Ci-PKA and a dimerized form of the HIB MATH domain (Flag-MATH-CC) (Fig. 3B) (44). On CK1 Regulates the Interaction Between Ci and HIB. HIB promotes Ci the other hand, CK1 RNAi enhanced the association between Myc- degradation by binding to both the N- and C-terminal regions of Ci Ci-PKA and Flag-MATH-CC (Fig. 3C), suggesting that CK1 inhibits through multivalent interactions with its MATH domain (44). To HIB binding to Ci. Furthermore, CK1* inhibited HIB binding to determine whether CK1 attenuates HIB-mediated degradation by both the N-terminal and C-terminal regions of Ci (Fig. S8), sug- regulating HIB/Ci interaction, we carried out coimmunoprecipita- gesting that CK1 regulates HIB binding to multiple Ci domains. tion experiments to determine whether HIB/Ci interaction is modulated by changes in CK1 activity. S2 cells were treated with CK1 Inhibits HIB Binding to Ci by Phosphorylating Multiple S/T-Rich a proteasome inhibitor, MG132, to stabilize the HIB/Ci complex Degrons. Both the N- and C-terminal regions of Ci contain before the immunoprecipitation assay. We found that the expres- a number of S/T-rich motifs that mediate HIB binding and Ci

E5654 | www.pnas.org/cgi/doi/10.1073/pnas.1416652111 Shi et al. Downloaded by guest on October 1, 2021 ABC be inhibited by kinases that phosphorylate HIB/SPOP degrons. PNAS PLUS We noticed that many HIB/SPOP degrons in Ci contain CK1 phosphorylation 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 by directly phosphorylating one or more HIB degrons. To test this hypothesis, we first examined whether CK1 phosphorylates CiA in vivo by monitoring the mobility shift of HA-Ci-PKA in S2 cells in the absence or presence of CK1* using D E the phospho-tag gel that specifically retards phosphorylated proteins (48). We found that coexpression of CK1* induced a mobility shift of HA-Ci-PKA but not HA-Cim1-6 (Fig. 3D), suggesting that CK1* stimulated Ci phosphorylation at HIB degrons. We also found that Hh induced a mobility shift of HA- Ci-PKA, which was abolished by CK1 RNAi (Fig. 3E, compare lanes 3 and 4 with lanes 1 and 2), suggesting that Hh stimulates FHCi-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 at HIB degrons. Taken together, these results suggest that CK1 phosphorylates one or more HIB degrons in vivo, which is stimulated by Hh. We then determined which HIB degron was phosphorylated GIby CK1 by applying an in vitro kinase assay in which GST-Ci fusion proteins containing individual HIB degrons (S1–S6 in Fig. 3 F and G) were incubated with a recombinant CK1 in the presence of γ-32p-ATP. We found that all six HIB degrons can be phosphorylated by CK1 in vitro, with S2 and S4 exhibiting the strongest and S6 exhibiting an intermediate level of phosphory- Fig. 3. CK1 promotes phosphorylation of Ci-PKA and inhibits recruitment of lation (Fig. 3G). We focused on S4 and S6 because our previous HIB. (A and B) Overexpression of CK1 inhibited HIB/Ci association. S2 cells study indicated that they are strong HIB-binding sites and reg- were transfected with Myc-Ci-PKA with or without the indicated HIB and CK1 ulate HIB-mediated Ci degradation in vivo (44). The putative expression constructs. After treatment with MG132 for 4 h, cell lysates were CK1 sites in S4 (S385,S387, and S388) and S6 (S1363,S1364, and subjected to immunoprecipitation and Western blot analysis using the in- S1365) were mutated to Ala to generate S4A and S6A. An in vitro dicated antibodies. Of note, both CK1α and CK1e are tagged by a Flag epi- e kinase assay indicated that GST-S4A no longer was phosphory- tope, and CK1 overlaps with a nonspecific band (asterisk) detected by the lated by CK1, and GST-S6A exhibited greatly reduced phos- anti-Flag antibody. (C) CK1 RNAi enhanced HIB/Ci association in response to Hh stimulation. S2 cells were treated with the control (luciferase) or CK1 α/e phorylation compared with GST-S6 (Fig. 3G). dsRNA before transfection with Myc-Ci-PKA and Flag-MATH-CC. The trans- To determine whether CK1-mediated phosphorylation of S4/6 fected cells were treated with or without Hh-conditioned medium, followed regulates HIB binding, we mutated CK1 sites in S4/6 to Asp (S4/6D) by immunoprecipitation and Western blot analysis using the indicated to mimic phosphorylation in the GST fusion proteins (GST-S4D antibodies. (D) CK1 induced phosphorylation of Ci-PKA but not Cim1-6. S2 and GST-S6D) or in the context of HA-Ci-PKA (HA-Ci-PKAS4D6D). cells were cotransfected with the indicated constructs and were treated with GST pull-down assays indicated that the phosphomimetic mutations MG132. Cell lysates were separated on Phos-tag–conjugated SDS/PAGE, abolished HIB binding to the corresponding degrons (Fig. 3H). followed by Western blot analysis with an anti-Myc antibody. (E) Hh stim- -PKA Furthermore, coimmunoprecipitation experiments revealed that ulated phosphorylation of Myc-Ci but not Myc-Cim1-6 through CK1. S2 HA-Ci-PKAS4D6D pulled down less Flag-MATH-CC than HA-Ci-PKA cells treated with luciferase or CK1α/e dsRNA were cotransfected with the indicated Ci constructs and were treated with or without Hh-conditioned (Fig. 3I), consistent with the notion that phosphorylation of S4/6 medium. After treatment with MG132 for 4 h, cell lysates were prepared and inhibits HIB/Ci interaction. separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. (F) A diagram of full-length Ci with six Phosphomimetic Ci Exhibits Delayed Degradation. To determine HIB-binding sites (S1–S6) indicated by individual bars and the sequences of whether CK1 protects Ci from HIB-mediated degradation by individual sites shown underneath. The S/T-rich sequences are underlined. phosphorylating the S/T-rich degrons, we first compared the sta- BIOLOGY (G) In vitro kinase assay using a recombinant CK1 and GST-Ci fusion proteins bility of HA-Ci-PKAS4D6D with that of HA-Ci-PKA in S2 cells treated DEVELOPMENTAL containing the indicated wild-type or mutated HIB-binding sites in the with Hh-conditioned medium. We found that HA-Ci-PKAS4D6D γ 32 presence of -[ P]ATP. (Left) Short (Top)orlong(Middle)exposureofthe exhibited increased stability compared with HA-Ci-PKA upon Hh autoradiograph is shown. (H) GST-Ci fusion proteins containing wild-type or -PKA – stimulation (Fig. 4 A and B). Unlike HA-Ci , which was desta- mutated S4 or S6 were incubated with cell extracts derived from HIB-N -PKAS4D6D expressing S2 cells. Input and bound HIB-N proteins were analyzed by Western bilized by CK1 RNAi, the stability of HA-Ci was not blot using an anti-HA antibody. (I) S2 cells were transfected with Flag-MATH- significantly affected by CK1 RNAi (Fig. 4 A and B), suggesting that -PKA CC alone or together with Myc-Ci-PKA or Myc-Ci-PKAS4D6D, followed by immu- phosphomimetic Ci is less dependent on CK1 for its durability noprecipitation and Western blot analysis using the indicated antibodies. Myc- in the presence of Hh. Ci-PKAS4D6D pulled down less Flag-MATH-CC than Myc-Ci-PKA. We next compared the stability of HA-Ci-PKAS4D6D with that of HA-Ci-PKA in wing imaginal discs. To ensure similar levels of transgene expression, we generated transformants for UAS-HA- degradation, and these S/T-rich degrons also are present in other Ci-PKA and UAS-HA-Ci-PKAS4D6D using the phiC31 integration HIB/SPOP substrates (44, 46). Interestingly, converting the S/T system in which the transgenes were inserted at the same genome residues of HIB/SPOP degrons with either phosphorylated resi- locus (49). We first expressed these transgenes in wing discs using dues or acidic residues to mimic phosphorylation blocked HIB/ aweakGal4driver,C765 (17) and found that HA-Ci-PKAS4D6D SPOP binding (44, 46), raising an interesting possibility that exhibited higher protein levels and induced higher levels of ectopic substrate recognition by Cul3HIB/SPOP family of E3 ligases could ptc expression than HA-Ci-PKA in P-compartment cells (Fig. 4 C–D′).

Shi et al. PNAS | Published online December 15, 2014 | E5655 Downloaded by guest on October 1, 2021 A B

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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 dsRNA were transfected with expression constructs for HA-Ci-PKA or HA-Ci-PKAS4D6D and Myc-CFP (as an internal control). After treatment with Hh-conditioned medium 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-Myc antibodies. 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 ± SD from 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′, F and F′, and H and H′) under the control of C765 (C–D′)orMS1096 (E–H′) in the absence (C–F′) or presence (G–H′)ofCRL were immunostained to show the expression 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′).

Of note, expression of HA-Ci-PKAS4D6D also induced weak ec- (44), raising the possibility that CK1 may play a conserved role in topic ptc expression in A-compartment cells (Fig. 4D′), consis- regulating Gli proteins. Consistent with this notion, we found -PKA tent with its being more stable than HA-Ci . It is likely that that CK1α RNAi reduced Gli-luc reporter gene expression overexpressed Ci was partially converted into a labile active form driven by a constitutively active form of Smo (SmoSD0-5) in in A-compartment cells away from the A/P boundary because of which all the CK1/GRK2 phosphorylation sites were converted the limiting amount of endogenous Sufu. to acidic residues (Fig. S9) (15), suggesting that CK1 has an When expressed under the control of MS1096,both -PKAS4D6D -PKA additional positive input in the Shh pathway downstream of Smo. HA-Ci and HA-Ci accumulated at high levels and To test the possibility that CK1 controls the stability of GliA, fully activated ptc expression in P-compartment cells (Fig. 4 E–F′). we took advantage of the observations that Gli proteins were However, when CRL was coexpressed with HA-Ci-PKA, both Ci Dro- protein level and ectopic ptc expression were diminished in controlled by Hh pathway components when expressed in P-compartment cells (Fig. 4 G and G′). In contrast, when CRL sophila (31, 52). Consistent with a previous finding (52), ex- > was coexpressed with HA-Ci-PKAS4D6D, a significant amount of pression of Myc-tagged Gli2 in wing discs (MS Myc-Gli2) HA-Ci-PKAS4D6D remained in P-compartment cells and induced induced ectopic ptc expression in P-compartment cells (Fig. 5 ectopic ptc expression in these cells (Fig. 4 H and H′). However, A–B′′). Coexpression of CRL diminished Myc-Gli2 levels and phosphomimetic mutations at S4/6 did not render CiA com- blocked Gli2-induced ectopic ptc expression in these cells (Fig. 5 pletely independent of CK1 in wing discs, because the levels of C–D′′), suggesting that CK1 inactivation resulted in the loss of HA-Ci-PKAS4D6D decreased in P-compartment cells expressing active Gli2. Coexpression of HIB-RNAi with CRL prevented Gli2 CRL (compare Fig. 4H with Fig. 4F). It is likely that CK1 can degradation and restored the ectopic expression of ptc in A regulate Ci stability by phosphorylating other HIB-degrons P-compartment cells (Fig. 5 E–F′′), suggesting that CK1 protects that are not altered in HA-Ci-PKAS4D6D. It is also possible that A active Gli2 from HIB-mediated degradation. CK1 phosphorylates additional target(s) to stabilize Ci . We then asked whether CK1 attenuates HIB/SPOP-mediated degradation of Gli2 by inhibiting its binding to Gli2 (44). CK1 Protects Gli2 from HIB/SPOP-Mediated Degradation. The task of Coexpression of Flag-SPOP with Myc-Gli2 in S2 cells diminished Ci in Hh signaling is divided between two members of the Gli family transcription factors in vertebrates, Gli2 and Gli3; Gli2 Myc-Gli2 protein levels (Fig. 5G) (44), which can be partially contributes mainly to the activator form (GliA) and Gli3 to the restored by coexpression of CK1* (Fig. 5G), suggesting that CK1 repressor form (GliR) of Gli activities (1, 50). Both full-length attenuates SPOP-mediated degradation of Gli2. In S2 cells Gli2 and Gli3 are subjected to HIB/SPOP-mediated degradation treated with MG132, expression of CK1* reduced the amounts in mammalian cultured cells as well as in Drosophila imaginal of Myc-Gli2 bound to Flag-SPOP or Flag-MATH-CC (Fig. 5 H discs (26, 31, 51). The degradation of Gli2/3 by HIB/SPOP is also and I), suggesting that CK1 inhibits the recognition of Gli2 by mediated by multiple S/T-rich degrons resembling those in Ci SPOP. Similar results were obtained with Gli3 (Fig. S10).

E5656 | www.pnas.org/cgi/doi/10.1073/pnas.1416652111 Shi et al. Downloaded by guest on October 1, 2021 A A’ A’’ B B’ B’’ PNAS PLUS

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Fig. 5. CK1 prevents HIB/SPOP-mediated down-regulation of Gli2 and promotes Hh signaling downstream of Sufu. (A–F′′) Late third-instar wing discs expressing MS > Myc-Gli2 (A–B′′), MS > Myc-Gli2 + CRL (C–D′′), or MS > 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 ptc expression in P-compartment cells were down-regulated by inactivation of CK1 (compare C–D′′ with A–B′′). Simultaneous inactivation of HIB and CK1 restored Myc-Gli2 protein levels and ectopic ptc expression in P-compartment cells (E–F′′). (G) CK1 inhibits SPOP-mediated down-regulation of Gli2. S2 cells were transfected 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 transfected with 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 normalized to Renilla luciferase activities. Combined expression of DN-CK1α and DN-CK1δ inhibited but overexpression of Flag-CK1α increased Gli-luc reporter gene −/− 5M α

expression. (K and L) Gli-luc reporter assay in Sufu MEFs transfected with Myc-Gli2 (K) or Myc-Gli2 (L), in the absence or presence of DN-CK1 and/or DN- BIOLOGY 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 three independent experiments. DEVELOPMENTAL

− − CK1 Acts Downstream of Sufu to Regulate GliA Activity. In Sufu activity in Sufu / MEFs; however, their combined expression mutant cells, GliA is constitutively produced independent of resulted in significant reduction of the Gli-luc activity, suggesting upstream signaling components (26, 53–55); therefore, we first that both CK1α and CK1δ/e are involved in preserving GliA − − − − tested whether CK1 regulates endogenous GliA activity in Sufu / activity in Sufu / MEFs. Consistent with notion, overexpression − − mouse embryonic fibroblasts (MEFs) using a Gli-luc reporter as- of CK1α in Sufu / MEFs increased Gli-luc activity (Fig. 5J). − − say. Consistent with previous findings (26, 53), Sufu / MEFs We also tested whether CK1 regulates GliA derived from − − exhibited high basal Gli-luc activity that was suppressed by exogenously expressed Gli2. Transfecting Sufu / MEFs with transfection with a mouse Sufu (mSufu) expression construct a Gli2 expression construct greatly increased Gli-luc activity (Fig. − − (Fig. 5J). To inactivate CK1, we transfected Sufu / MEFs with 5K). Coexpression of DN-CK1α and DN-CK1δ in combination dominant-negative forms of CK1α (DN-CK1α) and CK1δ (DN- suppressed but coexpression of wild-type CK1α increased Gli-luc CK1δ) either individually or in combination. A previous study activity induced by Gli2, suggesting that CK1 promotes Gli2A showed that DN-CK1α and DN-CK1δ selectively inhibit CK1α activity downstream of Sufu. Importantly, Gli-luc activity in- and CK1δ/e, respectively (56). As shown in Fig. 5J, DN-CK1α duced by Gli25m, a Gli2 variant containing substitutions in five slightly reduced but DN-CK1δ did not significantly alter Gli-luc S/T-rich degrons and resistant to SPOP-mediated degradation

Shi et al. PNAS | Published online December 15, 2014 | E5657 Downloaded by guest on October 1, 2021 (44), was not significantly affected by either gain or loss of A CK1 activity (Fig. 5L), further strengthening the notion that CK1 promotes GliA activity by attenuating SPOP-mediated degradation. Discussion The Ci/Gli family of transcription factors regulates animal de- velopment through the canonical Hh signaling pathway, and their activities are tightly controlled by different levels of Hh morphogen to elucidate distinct developmental outcomes. It has been well established that phosphorylation-mediated B of Ci/Gli plays an inhibitory role in Hh signaling by keeping the basal Hh pathway activity in check (11). Here we uncovered a previously 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 Hh stimulates CK1-mediated phosphorylation of Ci, likely at mul- tiple S/T-rich degrons, and that these phosphorylation events attenuate the recruitment of HIB/SPOP, thus slowing the Fig. 6. CK1 exerts both positive and negative roles in Hh signaling by HIB/SPOP A Cul3 -mediated degradation of Ci (Fig. 6). We propose phosphorylating multiple targets. (A) CK1 regulates Hh signaling at multiple that CK1-mediated phosphorylation of CiA increases its stability, levels. In the absence of Hh, CK1 phosphorylates Ci/Gli to promote Slimb/ allowing CiA levels to exceed critical thresholds to activate Hh βTRCP-mediated proteolytic processing that generates the repressor forms target genes. We also provide evidence that CK1 plays a con- of Ci/Gli (step 1). In Hh-stimulated cells, CK1 phosphorylates Smo to promote served role in the regulation of GliA. Ci/Gli activation (step 2) and protects the activated Ci/Gli from HIB/SPOP- mediated degradation (step 3). (B, Left) Under normal circumstances, CK1 CK1 was identified initially as a negative regulator of the Hh attenuates HIB/SPOP-mediated degradation of Ci/Gli, allowing CiA/GliA to signaling pathway that phosphorylates Ci at multiple sites fol- accumulate above certain thresholds necessary for the expression of Hh lowing the primed phosphorylation by PKA (35, 57). The se- target genes. (Right) When CK1 activity is reduced, CiA/GliA no longer is quential phosphorylation of Ci by PKA, CK1, and GSK3 recruits protected, leading to accelerated degradation of CiA/GliA by HIB/SPOP and β SCFSlimb/ TRCP that targets Ci for ubiquitin/proteasome-medi- premature loss of Hh pathway activity. See text for details. ated processing to generate CiR (Fig. 6A) (35, 58, 59). Later, several studies uncovered positive roles of CK1 in Hh signaling Previous studies suggested that CiA degradation is mediated in which CK1 phosphorylates and activates Smo and possibly Fu HIB/SPOP (14, 30, 39–41). Therefore, it was unexpected that inactivation of by the Cul3-based E3 ubiquitin ligase Cul3 and that CK1 compromised the Hh pathway activity elicited by constitu- Cul3/HIB-mediated degradation serves as a mechanism for ter- tively activated forms of Smo and Fu or by simultaneous in- minating Hh pathway activity, which is essential for normal activation of Cos2 and Sufu. To uncouple the positive role of Drosophila eye development (31, 32, 60, 61). HIB expression is CK1 in Hh signaling from its role in the regulation of Ci pro- up-regulated by Hh signaling in embryos as well as in imaginal cessing, we examined the consequences of CK1 inactivation on discs; thus, Cul3/HIB forms a negative feedback loop to fine- Hh signaling in P-compartment cells that do not express endog- tune Hh pathway activity in both embryonic and imaginal disk enous Ci but instead express an unprocessed form of Ci (Ci-PKA) development (31, 32). SPOP also is involved in the degradation from a transgene. We found that inactivation of CK1 blocked of active forms of Gli proteins, because removal of SPOP in Sufu Ci-PKA-induced ectopic ptc expression in P-compartment cells. It is mutant cells stabilized full-length Gli, leading to elevated Hh unlikely that the loss of ectopic ptc expression caused by CK1 pathway activity (26). Aside from the observation that HIB is up- HIB/SPOP RNAi results from the blockage of conversion of CiF into CiA regulated by Hh, it is not clear whether Cul3 -mediated because one would expect elevated levels of Ci-PKA if such were degradation of Ci/Gli is regulated by other mechanisms during the case. Instead, we observed diminished levels of Ci-PKA when development. Here, we demonstrate that CK1 counteracts HIB/SPOP A A CK1 was inactivated by CRL. Strikingly, coexpression of Fu-RNAi Cul3 -mediated degradation of Ci /Gli to prevent pre- with CRL restored Ci-PKA protein level but not ectopic ptc ex- mature loss of Hh signaling activity. Mechanistically, we showed pression. We interpret these results as showing that Ci-PKA was that CK1 attenuated binding of HIB/SPOP to Ci/Gli, likely by still converted into CiA in P-compartment cells expressing CRL, phosphorylating multiple S/T-rich degrons present in Ci/Gli2, likely because residual CK1 activity sufficed to activate Smo and although it remains possible that CK1 has additional target sites. Fu; however, CiA was degraded more rapidly when CK1 activity Interestingly, we found that Hh induced phosphorylation of was compromised, leading to a premature loss of Hh pathway Ci-PKA, which was abolished by CK1 RNAi. These phosphory- activity (Fig. 6B). In P-compartment cells coexpressing Fu-RNAi lation events are distinct from previously characterized PKA- and CRL,Ci-PKA no longer was converted into CiA because of the primed CK1 phosphorylation of Ci and appear to occur at complete loss of Fu activity and was accumulated in an inactive multiple S/T-rich degrons. Hence, Ci possesses two sets of CK1 form that did not rely on CK1 for its stability. Hence, CK1 is sites that play opposing roles in the Hh pathway and are regu- specifically required for the stabilization of CiA.Thisnotionwas lated by Hh signaling in the opposite directions: (i) PKA-primed confirmed by the cell-based assay in which we directly measured CK1 phosphorylation negatively regulates Ci activity by targeting whether inactivation of CK1 altered the stability of Ci-PKA.Our it for Slimb-mediated processing to generate CiR, and these results clearly showed that inactivation of CK1 shortened the half- phosphorylation events are inhibited by Hh; (ii) CK1-mediated life of Ci-PKA only in the presence of Hh signaling activity, sug- phosphorylation of Ci at multiple S/T-rich degrons preserves CiA gesting that CK1 activity is required to extend the lifetime of Hh- activity by attenuating HIB/SPOP-mediated degradation, and activated Ci. We found that CK1 RNAi reduced Ci staining in A- these phosphorylation events are stimulated by Hh (Fig. 6A). Of compartment cells in which both Cos2 and Sufu were inactivated, note, removing HIB in MS > CRL wing discs failed to rescue en suggesting that CK1 is required for the stabilization of CiA derived expression in A-compartment cells (Fig. S4), which requires the from endogenous Ci. highest levels of Hh pathway activity. One possible explanation is

E5658 | www.pnas.org/cgi/doi/10.1073/pnas.1416652111 Shi et al. Downloaded by guest on October 1, 2021 that upstream components such as Smo and Fu may not be fully and UAS-SmoSD (SmoSD123) (35, 39); UAS-Ci-PKA (42); UAS-SmoΔSAID (14); PNAS PLUS activated in these rescue experiments. Another possibility is that UAS-CC-FuEE (29); cos22 (65); UAS-HIB-RNAi (31); UAS-Cim1-6 and UAS- CK1 may positively regulate CiA activity through an additional MATH-CC (44); UAS-CK1α, UAS-CK1«,andUAS-CK1*(UAS-XCK1«-KD) (45); mechanism(s) independent of HIB. A recent study reported that UAS-Myc-Gli2 (52); UAS-Fu-RNAi (Vienna Drosophila Resource Center no. vertebrate Hh signaling conveys its gradient information by 27663); and UAS-Sufu-RNAi (Bloomington Drosophila Stock Center no. 28559). Amino acid substitutions of multiple S/T-rich degrons were generated by PCR- elaborately modulating multisite phosphorylation of Gli proteins based site-directed mutagenesis. UAS-HA-Ci-PKA and UAS-HA-Ci-PKAS4D6D were (62). Thus, it would be interesting to determine if Hh-induced inserted into the attP site at 71B as previously described (49). CK1 phosphorylation of Ci directly contributes its optimal transcriptional activity in addition to regulating its stability. Cell Culture, Luciferase Reporter Assay, Immunoprecipitation, Western Blot, How Hh does signaling differentially regulate these positive and Immunostaining. Drosophila S2 cells were cultured in Drosophila SFM and negative phosphorylation events? Previous studies revealed (Invitrogen) with 10% (vol/vol) FBS, 100 U/mL of penicillin, and 100 mg/ that Ci and its kinases, including PKA, GSK3, and CK1, form mL of streptomycin at 24 °C. Transfection was carried out using the Calcium protein complexes scaffolded by Cos2 and that Hh signaling Phosphate Transfection Kit (Specialty Media) according to the manu- induces either dissociation or composition change of these facturer’s instructions. Hh-conditioned medium was carried out as previously −/− complexes (22, 29, 63), thereby impeding PKA/GSK3/CK1- described (17). Sufu MEFs transfection and the Gli-luciferase reporter as- mediated Ci phosphorylation and proteolytic processing. It is say were carried out as described (66). Immunoprecipitation and Western thought that Ci/Gli forms a complex with Sufu in its inactive state blot analysis were carried out using standard protocols as previously de- scribed (22). The Phos tag-conjugated SDS/PAGE analysis was performed and that Hh activates Ci/Gli by dissociating it from Sufu, thus A A according to standard protocols (48). Phos tag-conjugated acrylamide was exposing Ci /Gli to HIB/SPOP and making it vulnerable for purchased from NARD Institute. Immunostaining of imaginal discs was car- ubiquitin/proteasome-mediated degradation (24, 29, 31). It is ried out as described (67). Antibodies used for this study were mouse anti- possible that an Hh-induced change in the formation or com- Flag (M2; Sigma), rabbit anti-Flag (Thermo Scientific), mouse anti-HA (F7; Santa position of Ci/Gli-Sufu complexes makes Ci/Gli more accessible Cruz), mouse anti-Myc (9E10; Santa Cruz), phospho-Fu (pT161/pT154) antibody to CK1, allowing CK1-mediated phosphorylation to counteract (29), Rat anti-Ci, 2A1 (68), rabbit anti-CKIe (kindly provided by D. M. Virshup, HIB/SPOP and modulate the speed of Ci/Gli degradation (Fig. 6B). Duke-NUS Graduate Medical School, Singapore), mouse anti-Ptc, and mouse This delicate balance may ensure appropriate levels of CiA/GliA for anti-En (Developmental Studies Hybridoma Bank). Hh signaling, and cells could change this balance to modulate Hh responses. For example, in Drosophila eye imaginal discs, RNAi in Drosophila S2 Cells. DNA templates corresponding to the coding α – e – differentiating cells posterior to the morphogenetic furrow up- regions of CK1 (nucleotides 601 1014) and CK1 (nucleotides 834 1323) regulate HIB to dampen the response to Hh by degrading Ci were generated by PCR and used to make dsRNA targeting the C terminus of CK1α and CK1e, respectively. dsRNA targeting the Firefly Luciferase coding (31, 32, 61). Our finding that the loss and gain of CK1 activity A A sequence was used as a control. dsRNAs were generated by MEGAscript can modulate the levels of Ci /Gli activity in opposite direc- High-Yield Transcription Kit (AM1334; Ambion). For the RNAi knockdown tions raises an interesting possibility that altering CK1 activity experiments, S2 cells first were cultured in serum-free medium containing may serve as a mechanism for fine-tuning Hh responses in dsRNA for 12 h at 24 °C. After FBS was added to a final concentration of certain contexts. 10% (vol/vol), dsRNA-treated cells were cultured overnight before trans- It has been well established that the Cul1-based E3 ubiquitin fection with DNA constructs. After additional culturing for 2 d, cells were ligase SCF complexes recognize substrates upon their phos- collected for analysis. phorylation, thus linking protein phosphorylation to protein degradation (64). Whether substrate recognition by other In Vitro Kinase Assay, GST Pull Down, and Protein Stability Assay. For the in Cullin families of E3 ligases also is regulated by phosphory- vitro kinase assay, individual GST-fusion proteins bound to glutathione beads 32 lation remains largely unknown. A previous study showed that were mixed with 0.1 mM ATP containing 10 mCi of γ- p-ATP and δ replacing the S/T residues in several SPOP degrons with phos- recombinant CK1 kinase (CK1 ; New England Biolabs) and were incubated at 30 °C for 1.5 h in reaction buffer [20 mM Tris·HCl (pH 8.0), 2 mM EDTA, phorylated residues blocked binding to SPOP in vitro (46), 10 mM MgCl2, 1 mM DTT]. Reactions were stopped by adding 4× SDS loading raising an interesting possibility that recognition of HIB/SPOP buffer, and the mixture was boiled at 100 °C for 5 min. The phosphorylated substrates could be regulated by kinases. Here we provide the GST-fusion proteins were analyzed by autoradiography after SDS/PAGE. The first evidence, to our knowledge, that substrate recognition by GST pull-down assay was carried out as described (44). The protein stability Cul3-based E3 ligases is negatively regulated by a kinase. Because assay was carried out as described (18). Cul3HIB/SPOP regulates a large family of proteins, our study raises HIB/SPOP an interesting possibility that the stability of other Cul3 ACKNOWLEDGMENTS. We thank Bing Wang for assistance, Drs. R. Holmgren, targets might also be regulated by phosphorylation. J. Wu, and P. T. Chuang and the Developmental Studies Hybridoma Bank for reagents, and the Vienna Drosophila Resource Center and Bloomington

Materials and Methods Drosophila Stock Center for fly stocks. This work was supported by National BIOLOGY Institutes of Health Grants GM061269 and GM067045 and Welch Founda- Drosophila Stocks and Transgenes. The following Drosophila stocks and tion Grant I-1603 (to J.J.), and National Science Foundation of China Grants DEVELOPMENTAL transgenes were used for this study: CRL, UAS-R*, UAS-SmoDN (Smo-PKA12), 31328017, 81322030, and 31271579 (to Y.C.).

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