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

Significance

Hedgehog (Hh) signaling controls development and tissue homeostasis through the Cubitus interruptus (Ci)/glioma-associated oncogene homolog (Gli) transcription factors, and abnormal Gli activity causes congenital diseases and cancers. Here we show that Ci/Gli phosphorylation by Casein kinase 1 positively regulates Hh pathway activity, providing insights into the regulation of Ci/Gli activity. By showing that phosphorylation protects the Ci/Gli activator from premature degradation, our study not only sheds lights on how the production and degradation of Ci/Gli activator are delicately balanced to achieve optimal pathway activity but also provides the first evidence (to our knowledge) that protein degradation by the Cullin 3 family of E3 ubiquitin ligases is negatively regulated by phosphorylation.

Abstract

Hedgehog (Hh) signaling governs many developmental processes by regulating the balance between the repressor (Ci^R/Gli^R) and activator (Ci^A/Gli^A) forms of Cubitus interruptus (Ci)/glioma-associated oncogene homolog (Gli) transcription factors. Although much is known about how Ci^R/Gli^R is controlled, the regulation of Ci^A/Gli^A remains poorly understood. Here we demonstrate that Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 and Suppressor of fused (Sufu) by protecting Ci^A from premature degradation. We show that Hh stimulates Ci phosphorylation by CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by the 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-mediated degradation of Ci^A, leading to premature loss of pathway activity. We also provide evidence that Gli^A is regulated by CK1 in a similar fashion and that CK1 acts downstream of Sufu to promote Sonic hedgehog signaling. Taken together, our study not only reveals an unanticipated and conserved mechanism by which phosphorylation of Ci/Gli positively regulates Hh signaling but also provides the first evidence, to our knowledge, that substrate recognition by the Cullin 3 family of E3 ubiquitin ligases is negatively regulated by a kinase.

The evolutionarily conserved Hedgehog (Hh) signaling pathway governs embryogenesis and adult tissue homeostasis by tightly controlling the balance between the repressor (Ci^R/Gli^R) and activator (Ci^A/Gli^A) forms of Cubitus interruptus (Ci)/Gli transcription factors (1–5). In Drosophila wing discs, Hh secreted from posterior (P) compartment cells moves into the anterior (A) compartment to form a local activity gradient near the A/P boundary. Low, intermediate, and peak levels of Hh differentially regulate the Ci^R/Ci^A ratio to activate decapentaplegic (dpp), patched (ptc), and engrailed (en), respectively (6–8). In humans, imbalance between Gli^R and Gli^A causes various birth defects and cancers (1, 9, 10).

Generation of Ci^R/Gli^R occurs in the absence of Hh. The kinesin-like proteins Costal2 (Cos2)/Kinesin superfamily member 7 (Kif7) and the tumor suppressor Suppressor of fused (Sufu) form protein complexes with full-length Ci/Gli (Ci^F/Gli^F) to prevent its nuclear localization and promote its phosphorylation by multiple kinases, including Protein kinase A (PKA), Casein kinase 1 (CK1), and Glycogen synthase kinase 3 (GSK3), which targets it for Supernumerary limbs (Slimb)/β-Transducin repeat containing E3 ubiquitin protein ligase (βTRCP)-mediated processing to generate truncated repressor forms (11). The production of Ci^A/Gli^A requires the binding of Hh ligand to the transmembrane receptor Ptc, which alleviates the inhibition of the transmembrane signal transducer Smoothened (Smo) by Ptc (1–3, 12, 13). Smo undergoes phosphorylation by multiple kinases that promote its active conformation and cell surface (Drosophila)/primary cilium (vertebrates) accumulation (11, 14–20). Smo-mediated intracellular signal transduction abrogates Ci/Gli processing into Ci^R/Gli^R and converts accumulated full-length Ci/Gli into Ci^A/Gli^A by dissociating Ci/Gli from Cos2/Kif7 and Sufu (8, 21–27). The Drosophila Ser/Thr kinase Fused (Fu) is required to antagonize Cos2- and Sufu-mediated inhibition of Ci (28–30), but its mammalian counterpart remains to be identified.

Ci^A is unstable and is degraded by the ubiquitin/proteasome pathway mediated by the MATH- and BTB-domain containing protein HIB (also called “Rdx”) (8, 31, 32). Interestingly, HIB is up-regulated in response to Hh in both embryos and imaginal discs (31, 32), and HIB also down-regulates Sufu through Crn (33), thus forming feedback loops to fine-tune Ci^A activity. However, it is not clear how HIB-mediated degradation of Ci^A is kept 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/Gli after PKA-primed phosphorylation, which is essential for the production of Ci^R/Gli^R (34–38); however, in the presence of Hh, CK1 phosphorylates Smo and likely Fu, to activate the Hh pathway (15, 30, 39–41). Here we uncover an unanticipated positive role of CK1 in the regulation of Ci^A downstream of Smo and Fu. We show that reduction in CK1 activity leads to destabilization of Ci^A and diminished Hh pathway activity. Mechanistically, we provide biochemical evidence that CK1 phosphorylates multiple Ser/Thr-rich degrons in Ci to attenuate HIB recognition and thus reduce the rate of HIB-mediated Ci^A degradation. Blockage of the HIB-mediated degradation either by inactivating HIB or by mutating the HIB degrons bypasses the requirement of CK1 in the stabilization of Ci^A. Importantly, we show that Gli^A is regulated by CK1 in a conserved manner and that CK1 positively regulates Gli activity in Sufu mutant cells.

Results

CK1 and PKA Differentially Regulate Ci Levels in Hh-Receiving Cells.

In wild-type wing discs of late third-instar larvae, Ci^F is accumulated in A-compartment cells near the A/P boundary because of the inhibition of Ci processing by Hh secreted from P-compartment cells (arrows in Fig. 1 A and B) (7); however, in A-compartment cells immediately adjacent to the A/P boundary that receive high levels of Hh, Ci staining is diminished because of the conversion of Ci^F into labile Ci^A (arrowheads in Fig. 1 A and B) (8), as evidenced by the expression of the high-threshold Hh target gene en in these cells (arrowhead in Fig. 1B′).

Fig. 1.

CK1 positively regulates Hh signaling at the level of Ci. (A–F′) Late third-instar wild-type (control) wing discs or wing discs expressing the indicated 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 right. The A compartment is marked by the expression of Ci. In control wing discs, Ci is accumulated 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' indicates en expression in A-compartment cells. CK1 RNAi resulted in down-regulation of Ci in A-compartment cells near the A/P boundary (arrowheads in C and D). Fu RNAi or expression of Smo^DN 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 expression 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 show the expression of Ci (red), Ptc (green), and En (blue). Sufu RNAi in cos2 mutant discs induced ectopic en expression but down-regulated the Ci protein level (arrowheads in G, H, and H'). Combined RNAi of CK1 and Sufu in cos2 mutant discs blocked en expression in A-compartment cells (arrowhead in J′) and further reduced the Ci protein level (arrowheads in I and J). Dashed lines demarcate the A/P boundary (B, B′, D, and D′).

Inactivation of CK1 using a wing-specific Gal4 driver MS1096 to express CRL (MS > CRL), a UAS-CK1-RNAi transgene that knocks down both CK1α and CK1ε (35), ectopically stabilized Ci^F in A-compartment cells distant from the A/P boundary (arrows in Fig. 1 C and D), consistent with previous findings that phosphorylation of Ci^F by CK1 is required for its proteolytic processing (35). MS > CRL also reduced ptc expression at the A/P boundary and blocked Hh-dependent en expression in A-compartment cells (Fig. 1 C′ and D′), as is consistent with CK1 having a positive role in Smo/Fu activation (30, 39–41). Similar results were obtained when PKA was inactivated by expressing a 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 expression in 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 compromised Smo/Fu activation and consequent failure of Ci^A production. However, we noticed that Ci levels in A-compartment cells near the A/P boundary were much lower than in A-compartment cells away from the boundary (arrowheads in Fig. 1 C and D), a result that would not be expected if MS > CRL blocked both Ci processing and Ci^F-to-Ci^A conversion. This finding is in sharp contrast to the nearly uniform accumulation of Ci in A-compartment cells of MS > R* wing discs (Fig. S1), a phenotype expected with the blockage of both Ci processing and Ci^A production. Thus, the mechanism(s) by which CRL diminishes Hh signaling activity may differ from the mechanism(s) by which PKA is inactivated. Of note, coexpressing either CK1α or CK1ε blocked CRL-mediated down-regulation of Ci in A-compartment cells near the A/P boundary and rescued ptc and en expression (Fig. S1), confirming that the effect of CRL on Ci level and activity results from the loss of CK1α/ε activity.

That the destabilization of Ci by CRL occurs strictly in A-compartment cells near the A/P boundary implies that this process is Hh-dependent. Therefore, it is possible that MS > CRL only attenuated but did not completely block Smo/Fu activation because of incomplete CK1 knockdown and that the remaining Smo/Fu activity still could convert significant amounts of Ci^F into Ci^A, leading to reduced Ci levels at the A/P boundary. When CK1 activity was compromised, Ci^A either was less active or was lost prematurely (see below), leading to diminished Hh pathway activity. Consistent with this notion, coexpression of Fu-RNAi or a dominant-negative form of Smo (Smo^DN) with CRL abolished Hh-induced ptc expression but stabilized Ci^F at the A/P boundary (arrowheads in Fig. 1 E–F′), indicating that Smo and Fu were still activated, at least partially, to convert Ci^F into labile Ci^A in MS > CRL wing discs. Taken together, these results imply that CK1 may have additional positive role(s) in the Hh pathway downstream of Smo/Fu.

CK1 Positively Regulates Ci Stability and Activity Downstream of Cos2 and Sufu.

To determine whether CK1 could exert a positive role downstream of Smo/Fu, we first examined if CK1 is required for optimal pathway activation elicited by constitutively active Smo and Fu. Expression of SmoSD (a Smo variant activated by converting PKA/CK1 phosphorylation clusters into acidic residues), SmoΔSAID (a Smo variant activated by deleting its auto-inhibitory domain), or CC-FuEE (a Fu variant activated by forced dimerization in combination with phosphomimetic mutations in its kinase activation loop) using MS1096 induced ectopic expression of all Hh target genes including ptc and en in A-compartment cells (Fig. S2 A and A′, C and C′, and E and E′) (14, 23, 39); however, the ectopic en expression was blocked by coexpression of CRL (compare Fig. S2 B′, D′, F′with Fig. S2 A′, C′, and E′) (30), raising the possibility that CK1 is required to sustain Hh signaling downstream of these active forms of Smo/Fu.

To test the possibility that CK1 positively regulates Hh signaling downstream of Smo/Fu, we examined whether CK1 is required for Hh pathway activity in wing discs in which both Cos2 and Sufu were inactivated so that Ci was constitutively active independent of Smo/Fu activation (1, 12). In cos2 mutant wing discs, Ci was accumulated uniformly in A-compartment cells because Cos2 is required for Ci phosphorylation and proteolytic processing (22); ptc was ectopically expressed in A-compartment cells at low levels because of accumulated Ci^F, but A-compartment en expression was lost because of compromised Fu activation (29, 43). Expression of Sufu-RNAi using MS1096 in cos2 mutant wing discs (referred to as “cos2 Sufu double-mutant discs”) resulted in increased expression of ptc (Fig. 1G′), ectopic expression of en (arrowhead in Fig. 1H′), and concomitant reduction of Ci staining in A-compartment cells (arrowheads in Fig. 1 G and H) because of the conversion of Ci^F into labile Ci^A in the absence of Sufu (8, 43). CRL blocked ectopic en expression and reduced ptc expression in cos2 Sufu double-mutant discs (Fig. 1 I′ and J′). In addition, CRL further reduced endogenous Ci levels in cos2 Sufu double-mutant discs (compare Fig. 1 I and J with Fig. 1 G and H). Because conversion of Ci^F to Ci^A in the cos2 Sufu double-mutant background is independent of upstream signaling, diminished Hh pathway activity and reduced Ci staining by CRL in this condition were likely caused by the premature loss of Ci^A.

CK1 Regulates the Stability of Ci^A.

To test further the possibility that CK1 protects Ci^A from premature loss, we expressed a processing-resistant form of Ci with three PKA sites (S838, S856, and S892) mutated to Ala (Ci^-PKA) (42) in wing discs either alone or together with CRL using MS1096. Consistent with our previous findings, expression of Ci^-PKA in P-compartment cells activated high levels of ptc expression (arrowhead in Fig. 2A′), suggesting that Ci^-PKA was converted into Ci^A by Hh. Strikingly, CRL dramatically decreased the levels of Ci^-PKA as well as ptc expression in P-compartment cells (compare Fig. 2 B and B′ with Fig. 2 A and A′), suggesting that CK1 is required for the maintenance of activated Ci^-PKA.

Fig. 2.

CK1 protects Ci^A from HIB-mediated degradation. (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 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 ectopic ptc expression (arrowheads in C and C′), whereas combined knockdown of HIB and CK1 restored 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 control (luciferase) dsRNA (G and I), CK1α/ε dsRNA (H and J), HIB dsRNA (K), or CK1α/ε + 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 subject to Western blot analysis with anti-HA and anti-Myc antibodies. Myc-CFP was used as an internal control. Quantification of HA-Ci^-PKA levels at different time points is shown below each autoradiogram. Data are means ± SD from three independent experiments.

Consistent with Fu being activated to generate labile Ci^A in MS > CRL wing discs, coexpression of Fu-RNAi with CRL completely blocked ptc expression but elevated Ci^-PKA levels in P-compartment cells (Fig. 2 C and C′). The accumulation of Ci^-PKA in the P-compartment cells when both CK1 and Fu were inactivated suggests that CK1 activity is not required for the stabilization of inactive Ci^-PKA. We found that expression of either CK1α or CK1ε in CRL-expressing P-compartment cells rescued ptc expression and restored Ci^-PKA levels (Fig. S3), consistent with the notion that both CK1 isoforms can promote Ci^A stabilization.

CK1 Protects Ci^A from HIB-Mediated Degradation.

We have shown previously that Ci^A degradation is mediated by the Cullin 3 (Cul3)-based E3 ubiquitination ligase that contains HIB (31). To determine whether the loss of Ci^A in CRL-expressing P-compartment cells is caused by the accelerated degradation by HIB, we coexpressed HIB-RNAi and CRL with Ci^-PKA. We found that HIB inactivation restored Ci^-PKA levels in CRL-expressing P-compartment cells (compare Fig. 2D with Fig. 2B). Unlike Fu RNAi, which stabilized inactive Ci^-PKA (Fig. 2 C and C′), HIB RNAi stabilized Ci^-PKA in an active form, as indicated by the ectopic expression of ptc (Fig. 2D′). An HIB-binding–deficient form of Ci (Cim1-6) (44) remained stable and induced ectopic ptc expression in CRL-expressing P-compartment cells (Fig. 2 E–F′). Furthermore, we found that combined knockdown of HIB and CK1 restored endogenous Ci and ptc expression in A-compartment cells near the A/P boundary but failed to rescue CRL-suppressed en expression in A-compartment cells (Fig. S4), implying that CK1 may regulate Hh pathway activity positively through an additional mechanism(s) that is independent of HIB. Taken together, these results suggest that CK1 is dispensable for Ci^A stability when HIB-mediated degradation is blocked.

We next determined whether gain of CK1 function could stabilize Ci^A. When expressed alone, Ci^-PKA was down-regulated in P-compartment cells because of its conversion into labile Ci^A (Fig. S5). Indeed, inactivation of HIB by RNAi abolished this down-regulation, allowing Ci^-PKA to accumulate at levels similar to those in A-compartment cells (Fig. S5). Overexpression of either CK1α or CK1ε also stabilized Ci^-PKA in P-compartment cells (Fig. S5). Furthermore, overexpression of CK1 in the posterior region of eye imaginal discs using the GMR Gal4 driver increased the levels of endogenous Ci (Fig. S5), phenocopying HIB RNAi in these cells (Fig. S5) (31) and suggesting that up-regulation of CK1 activity could counteract HIB-mediated degradation of Ci.

To test directly whether CK1 regulates the stability of activated Ci, we used a cell-based assay in which we measured the stability of Ci^-PKA under Hh-stimulated and unstimulated conditions. S2 cells were transfected with HA-tagged Ci^-PKA together with Myc-CFP as an internal control. The cells were treated with Hh-conditioned medium or control medium as well as with CK1α/ε dsRNA or luciferase (Luc) dsRNA as a control. Under these conditions, CK1 RNAi did not block Fu activation because Hh stimulated Fu phosphorylation in the presence of CK1α/ε dsRNA (Fig. S6). After cells were treated with cycloheximide (CHX) to block protein synthesis, HA-Ci^-PKA protein levels 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 the stability of HA-Ci^-PKA in the presence of Hh (compare Fig. 2K with Fig. 2I), consistent with the notion that Hh converts Ci^F into labile Ci^A degraded by HIB. On the other hand, CK1 RNAi further destabilized HA-Ci^-PKA in the presence of Hh (compare Fig. 2J with Fig. 2I) but did not affect the stability of HA-Ci^-PKA in the absence of Hh (Fig. 2H). Finally, inactivation of HIB restored the stability of HA-Ci^-PKA in the presence of both Hh and CK1α/ε dsRNA (compare Fig. 2L with Fig. 2J). Knockdown efficiency for individual dsRNAs was confirmed by Western blot analysis of epitope-tagged transgene expression (Fig. S7). Taken together, these results strengthen the conclusion derived from in vivo experiments that CK1 protects Hh-activated Ci by antagonizing HIB-mediated degradation.

CK1 Regulates the Interaction Between Ci and HIB.

HIB promotes Ci degradation by binding to both the N- and C-terminal regions of Ci through multivalent interactions with its MATH domain (44). To determine whether CK1 attenuates HIB-mediated degradation by regulating HIB/Ci interaction, we carried out coimmunoprecipitation experiments to determine whether HIB/Ci interaction is modulated by changes in CK1 activity. S2 cells were treated with a proteasome inhibitor, MG132, to stabilize the HIB/Ci complex before the immunoprecipitation assay. We found that the expression of Flag-CK1α, Flag-CK1ε, or both reduced the amounts of HA-HIB coimmunoprecipitated with Myc-Ci^-PKA (Fig. 3A). Furthermore, expression of the kinase domain of Xenopus CK1ε (referred to as “CK1*”), which exhibits potent activity in vivo (45), also inhibited the interaction between Myc-Ci^-PKA and a dimerized form of the HIB MATH domain (Flag-MATH-CC) (Fig. 3B) (44). On the other hand, CK1 RNAi enhanced the association between Myc-Ci^-PKA and Flag-MATH-CC (Fig. 3C), suggesting that CK1 inhibits HIB binding to Ci. Furthermore, CK1* inhibited HIB binding to both the N-terminal and C-terminal regions of Ci (Fig. S8), suggesting that CK1 regulates HIB binding to multiple Ci domains.

Fig. 3.

CK1 promotes phosphorylation of Ci^-PKA and inhibits recruitment of HIB. (A and B) Overexpression of CK1 inhibited HIB/Ci association. S2 cells were transfected with Myc-Ci^-PKA with or without the indicated HIB and CK1 expression constructs. After treatment with MG132 for 4 h, cell lysates were subjected to immunoprecipitation and Western blot analysis using the indicated antibodies. Of note, both CK1α and CK1ε are tagged by a Flag epitope, and CK1ε overlaps with a nonspecific band (asterisk) detected by the 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/ε dsRNA before transfection with Myc-Ci^-PKA and Flag-MATH-CC. The transfected cells were treated with or without Hh-conditioned medium, followed by immunoprecipitation and Western blot analysis using the indicated antibodies. (D) CK1 induced phosphorylation of Ci^-PKA but not Cim1-6. S2 cells were cotransfected with the indicated constructs and were treated with MG132. Cell lysates were separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. (E) Hh stimulated phosphorylation of Myc-Ci^-PKA but not Myc-Cim1-6 through CK1. S2 cells treated with luciferase or CK1α/ε dsRNA were cotransfected with the indicated Ci constructs and were treated with or without Hh-conditioned medium. After treatment with MG132 for 4 h, cell lysates were prepared and 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 HIB-binding sites (S1–S6) indicated by individual bars and the sequences of individual sites shown underneath. The S/T-rich sequences are underlined. (G) In vitro kinase assay using a recombinant CK1 and GST-Ci fusion proteins containing the indicated wild-type or mutated HIB-binding sites in the presence of γ-[³²P]ATP. (Left) Short (Top) or long (Middle) exposure of the autoradiograph is shown. (H) GST-Ci fusion proteins containing wild-type or mutated S4 or S6 were incubated with cell extracts derived from HIB-N–expressing S2 cells. Input and bound HIB-N proteins were analyzed by Western blot 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 immunoprecipitation and Western blot analysis using the indicated antibodies. Myc-Ci^-PKAS4D6D pulled down less Flag-MATH-CC than Myc-Ci^-PKA.

CK1 Inhibits HIB Binding to Ci by Phosphorylating Multiple S/T-Rich Degrons.

Both the N- and C-terminal regions of Ci contain a number of S/T-rich motifs that mediate HIB binding and Ci degradation, and these S/T-rich degrons also are present in other HIB/SPOP substrates (44, 46). Interestingly, converting the S/T residues of HIB/SPOP degrons with either phosphorylated residues or acidic residues to mimic phosphorylation blocked HIB/SPOP binding (44, 46), raising an interesting possibility that substrate recognition by Cul3^HIB/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 CK1 phosphorylation consensus sites: D/E/S(P)/T(P)[X1-3]S/T (boldface 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 Ci^A in vivo by monitoring the mobility shift of HA-Ci^-PKA in S2 cells in the absence or presence of CK1* using 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 Ci^-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 by 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 phosphorylation (Fig. 3G). We focused on S4 and S6 because our previous study indicated that they are strong HIB-binding sites and regulate HIB-mediated Ci degradation in vivo (44). The putative CK1 sites in S4 (S₃₈₅, S₃₈₇, and S₃₈₈) and S6 (S₁₃₆₃, S₁₃₆₄, and S₁₃₆₅) were mutated to Ala to generate S4A and S6A. An in vitro kinase assay indicated that GST-S4A no longer was phosphorylated by CK1, and GST-S6A exhibited greatly reduced phosphorylation 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-S4D and GST-S6D) or in the context of HA-Ci^-PKA (HA-Ci^-PKAS4D6D). GST pull-down assays indicated that the phosphomimetic mutations abolished HIB binding to the corresponding degrons (Fig. 3H). Furthermore, coimmunoprecipitation experiments revealed that HA-Ci^-PKAS4D6D pulled down less Flag-MATH-CC than HA-Ci^-PKA (Fig. 3I), consistent with the notion that phosphorylation of S4/6 inhibits HIB/Ci interaction.

Phosphomimetic Ci Exhibits Delayed Degradation.

To determine whether CK1 protects Ci from HIB-mediated degradation by phosphorylating the S/T-rich degrons, we first compared the stability of HA-Ci^-PKAS4D6D with that of HA-Ci^-PKA in S2 cells treated with Hh-conditioned medium. We found that HA-Ci^-PKAS4D6D exhibited increased stability compared with HA-Ci^-PKA upon Hh stimulation (Fig. 4 A and B). Unlike HA-Ci^-PKA, which was destabilized by CK1 RNAi, the stability of HA-Ci^-PKAS4D6D was not significantly affected by CK1 RNAi (Fig. 4 A and B), suggesting that phosphomimetic Ci^-PKA is less dependent on CK1 for its durability in the presence of Hh.

Fig. 4.

Phosphomimetic Ci exhibits increased stability. (A) Protein stability assays for Ci^-PKA and Ci^-PKAS4D6D. S2 cells treated with or without CK1α/ε 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′) or MS1096 (E–H′) in the absence (C–F′) or presence (G–H′) of CRL 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′).

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-Ci^-PKA and UAS-HA-Ci^-PKAS4D6D using the phiC31 integration system in which the transgenes were inserted at the same genome locus (49). We first expressed these transgenes in wing discs using a weak Gal4 driver, C765 (17) and found that HA-Ci^-PKAS4D6D exhibited higher protein levels and induced higher levels of ectopic ptc expression than HA-Ci^-PKA in P-compartment cells (Fig. 4 C–D′). Of note, expression of HA-Ci^-PKAS4D6D also induced weak ectopic ptc expression in A-compartment cells (Fig. 4D′), consistent with its being more stable than HA-Ci^-PKA. It is likely that overexpressed Ci was partially converted into a labile active form in A-compartment cells away from the A/P boundary because of the limiting amount of endogenous Sufu.

When expressed under the control of MS1096, both HA-Ci^-PKAS4D6D and HA-Ci^-PKA accumulated at high levels and fully activated ptc expression in P-compartment cells (Fig. 4 E–F′). However, when CRL was coexpressed with HA-Ci^-PKA, both Ci protein level and ectopic ptc expression were diminished in P-compartment cells (Fig. 4 G and G′). In contrast, when CRL was coexpressed with HA-Ci^-PKAS4D6D, a significant amount of HA-Ci^-PKAS4D6D remained in P-compartment cells and induced ectopic ptc expression in these cells (Fig. 4 H and H′). However, phosphomimetic mutations at S4/6 did not render Ci^A completely independent of CK1 in wing discs, because the levels of HA-Ci^-PKAS4D6D decreased in P-compartment cells expressing CRL (compare Fig. 4H with Fig. 4F). It is likely that CK1 can regulate Ci^A stability by phosphorylating other HIB-degrons that are not altered in HA-Ci^-PKAS4D6D. It is also possible that CK1 phosphorylates additional target(s) to stabilize Ci^A.

CK1 Protects Gli2 from HIB/SPOP-Mediated Degradation.

The task of Ci in Hh signaling is divided between two members of the Gli family transcription factors in vertebrates, Gli2 and Gli3; Gli2 contributes mainly to the activator form (Gli^A) and Gli3 to the repressor form (Gli^R) of Gli activities (1, 50). Both full-length Gli2 and Gli3 are subjected to HIB/SPOP-mediated degradation in mammalian cultured cells as well as in Drosophila imaginal discs (26, 31, 51). The degradation of Gli2/3 by HIB/SPOP is also mediated by multiple S/T-rich degrons resembling those in Ci (44), raising the possibility that CK1 may play a conserved role in regulating Gli proteins. Consistent with this notion, we found that CK1α RNAi reduced Gli-luc reporter gene expression driven by a constitutively active form of Smo (SmoSD0-5) in which all the CK1/GRK2 phosphorylation sites were converted to acidic residues (Fig. S9) (15), suggesting that CK1 has an additional positive input in the Shh pathway downstream of Smo.

To test the possibility that CK1 controls the stability of Gli^A, we took advantage of the observations that Gli proteins were controlled by Hh pathway components when expressed in Drosophila (31, 52). Consistent with a previous finding (52), expression of Myc-tagged Gli2 in wing discs (MS > Myc-Gli2) induced ectopic ptc expression in P-compartment cells (Fig. 5 A–B′′). Coexpression of CRL diminished Myc-Gli2 levels and blocked Gli2-induced ectopic ptc expression in these cells (Fig. 5 C–D′′), suggesting that CK1 inactivation resulted in the loss of active Gli2. Coexpression of HIB-RNAi with CRL prevented Gli2 degradation and restored the ectopic expression of ptc in P-compartment cells (Fig. 5 E–F′′), suggesting that CK1 protects active Gli2 from HIB-mediated degradation.

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 indicated 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 expression. (K and L) Gli-luc reporter assay in Sufu^−/− MEFs transfected with Myc-Gli2 (K) or Myc-Gli2^5M (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-Gli2^5m was influenced by changing CK1 activity. Data are means ± SD from three independent experiments.

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 diminished Myc-Gli2 protein levels (Fig. 5G) (44), which can be partially restored by coexpression of CK1* (Fig. 5G), suggesting that CK1 attenuates SPOP-mediated degradation of Gli2. In S2 cells treated with MG132, expression of CK1* reduced the amounts of Myc-Gli2 bound to Flag-SPOP or Flag-MATH-CC (Fig. 5 H and I), suggesting that CK1 inhibits the recognition of Gli2 by SPOP. Similar results were obtained with Gli3 (Fig. S10).

CK1 Acts Downstream of Sufu to Regulate Gli^A Activity.

In Sufu mutant cells, Gli^A is constitutively produced independent of upstream signaling components (26, 53–55); therefore, we first tested whether CK1 regulates endogenous Gli^A activity in Sufu^−/− mouse embryonic fibroblasts (MEFs) using a Gli-luc reporter assay. Consistent with previous findings (26, 53), Sufu^−/− MEFs exhibited high basal Gli-luc activity that was suppressed by transfection with a mouse Sufu (mSufu) expression construct (Fig. 5J). To inactivate CK1, we transfected Sufu^−/− MEFs with dominant-negative forms of CK1α (DN-CK1α) and CK1δ (DN-CK1δ) either individually or in combination. A previous study showed that DN-CK1α and DN-CK1δ selectively inhibit CK1α and CK1δ/ε, respectively (56). As shown in Fig. 5J, DN-CK1α slightly reduced but DN-CK1δ did not significantly alter Gli-luc activity in Sufu^−/− MEFs; however, their combined expression resulted in significant reduction of the Gli-luc activity, suggesting that both CK1α and CK1δ/ε are involved in preserving Gli^A activity in Sufu^−/− MEFs. Consistent with notion, overexpression of CK1α in Sufu^−/− MEFs increased Gli-luc activity (Fig. 5J).

We also tested whether CK1 regulates Gli^A derived from exogenously expressed Gli2. Transfecting Sufu^−/− MEFs with a Gli2 expression construct greatly increased Gli-luc activity (Fig. 5K). Coexpression of DN-CK1α and DN-CK1δ in combination suppressed but coexpression of wild-type CK1α increased Gli-luc activity induced by Gli2, suggesting that CK1 promotes Gli2^A activity downstream of Sufu. Importantly, Gli-luc activity induced by Gli2^5m, a Gli2 variant containing substitutions in five S/T-rich degrons and resistant to SPOP-mediated degradation (44), was not significantly affected by either gain or loss of CK1 activity (Fig. 5L), further strengthening the notion that CK1 promotes Gli^A activity by attenuating SPOP-mediated degradation.

Discussion

The Ci/Gli family of transcription factors regulates animal development 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 proteolysis 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 regulation of Ci/Gli activator activity, i.e., the protection of Ci^A/Gli^A from premature degradation. We provide evidence that Hh stimulates CK1-mediated phosphorylation of Ci, likely at multiple S/T-rich degrons, and that these phosphorylation events attenuate the recruitment of HIB/SPOP, thus slowing the Cul3^HIB/SPOP-mediated degradation of Ci^A (Fig. 6). We propose that CK1-mediated phosphorylation of Ci^A increases its stability, allowing Ci^A levels to exceed critical thresholds to activate Hh target genes. We also provide evidence that CK1 plays a conserved role in the regulation of Gli^A.

Fig. 6.

CK1 exerts both positive and negative roles in Hh signaling by phosphorylating multiple targets. (A) CK1 regulates Hh signaling at multiple levels. In the absence of Hh, CK1 phosphorylates Ci/Gli to promote Slimb/βTRCP-mediated proteolytic processing that generates the repressor forms of Ci/Gli (step 1). In Hh-stimulated cells, CK1 phosphorylates Smo to promote Ci/Gli activation (step 2) and protects the activated Ci/Gli from HIB/SPOP-mediated degradation (step 3). (B, Left) Under normal circumstances, CK1 attenuates HIB/SPOP-mediated degradation of Ci/Gli, allowing Ci^A/Gli^A to accumulate above certain thresholds necessary for the expression of Hh target genes. (Right) When CK1 activity is reduced, Ci^A/Gli^A no longer is protected, leading to accelerated degradation of Ci^A/Gli^A by HIB/SPOP and premature loss of Hh pathway activity. See text for details.

CK1 was identified initially as a negative regulator of the Hh signaling pathway that phosphorylates Ci at multiple sites following the primed phosphorylation by PKA (35, 57). The sequential phosphorylation of Ci by PKA, CK1, and GSK3 recruits SCF^Slimb/βTRCP that targets Ci for ubiquitin/proteasome-mediated processing to generate Ci^R (Fig. 6A) (35, 58, 59). Later, several studies uncovered positive roles of CK1 in Hh signaling in which CK1 phosphorylates and activates Smo and possibly Fu (14, 30, 39–41). Therefore, it was unexpected that inactivation of CK1 compromised the Hh pathway activity elicited by constitutively activated forms of Smo and Fu or by simultaneous inactivation of Cos2 and Sufu. To uncouple the positive role of CK1 in Hh signaling from its role in the regulation of Ci processing, we examined the consequences of CK1 inactivation on Hh signaling in P-compartment cells that do not express endogenous Ci but instead express an unprocessed form of Ci (Ci^-PKA) from a transgene. We found that inactivation of CK1 blocked Ci^-PKA-induced ectopic ptc expression in P-compartment cells. It is unlikely that the loss of ectopic ptc expression caused by CK1 RNAi results from the blockage of conversion of Ci^F into Ci^A because one would expect elevated levels of Ci^-PKA if such were the case. Instead, we observed diminished levels of Ci^-PKA when CK1 was inactivated by CRL. Strikingly, coexpression of Fu-RNAi with CRL restored Ci^-PKA protein level but not ectopic ptc expression. We interpret these results as showing that Ci^-PKA was still converted into Ci^A in P-compartment cells expressing CRL, likely because residual CK1 activity sufficed to activate Smo and Fu; however, Ci^A was degraded more rapidly when CK1 activity was compromised, leading to a premature loss of Hh pathway activity (Fig. 6B). In P-compartment cells coexpressing Fu-RNAi and CRL, Ci^-PKA no longer was converted into Ci^A because of the complete loss of Fu activity and was accumulated in an inactive form that did not rely on CK1 for its stability. Hence, CK1 is specifically required for the stabilization of Ci^A. This notion was confirmed by the cell-based assay in which we directly measured whether inactivation of CK1 altered the stability of Ci^-PKA. Our results clearly showed that inactivation of CK1 shortened the half-life of Ci^-PKA only in the presence of Hh signaling activity, suggesting 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 Ci^A derived from endogenous Ci.

Previous studies suggested that Ci^A degradation is mediated by the Cul3-based E3 ubiquitin ligase Cul3^HIB/SPOP and that Cul3/HIB-mediated degradation serves as a mechanism for terminating Hh pathway activity, which is essential for normal Drosophila eye development (31, 32, 60, 61). HIB expression is up-regulated by Hh signaling in embryos as well as in imaginal discs; thus, Cul3/HIB forms a negative feedback loop to fine-tune Hh pathway activity in both embryonic and imaginal disk development (31, 32). SPOP also is involved in the degradation of active forms of Gli proteins, because removal of SPOP in Sufu mutant cells stabilized full-length Gli, leading to elevated Hh pathway activity (26). Aside from the observation that HIB is up-regulated by Hh, it is not clear whether Cul3^HIB/SPOP-mediated degradation of Ci/Gli is regulated by other mechanisms during development. Here, we demonstrate that CK1 counteracts Cul3^HIB/SPOP-mediated degradation of Ci^A/Gli^A to prevent premature loss of Hh signaling activity. Mechanistically, we showed that CK1 attenuated binding of HIB/SPOP to Ci/Gli, likely by phosphorylating 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 of Ci^-PKA, which was abolished by CK1 RNAi. These phosphorylation events are distinct from previously characterized PKA-primed CK1 phosphorylation of Ci and appear to occur at multiple S/T-rich degrons. Hence, Ci possesses two sets of CK1 sites that play opposing roles in the Hh pathway and are regulated by Hh signaling in the opposite directions: (i) PKA-primed CK1 phosphorylation negatively regulates Ci activity by targeting it for Slimb-mediated processing to generate Ci^R, and these phosphorylation events are inhibited by Hh; (ii) CK1-mediated phosphorylation of Ci at multiple S/T-rich degrons preserves Ci^A activity by attenuating HIB/SPOP-mediated degradation, and these phosphorylation events are stimulated by Hh (Fig. 6A). Of note, removing HIB in MS > CRL wing discs failed to rescue en expression in A-compartment cells (Fig. S4), which requires the highest levels of Hh pathway activity. One possible explanation is that upstream components such as Smo and Fu may not be fully activated in these rescue experiments. Another possibility is that CK1 may positively regulate Ci^A activity through an additional mechanism(s) independent of HIB. A recent study reported that vertebrate Hh signaling conveys its gradient information by elaborately modulating multisite phosphorylation of Gli proteins (62). Thus, it would be interesting to determine if Hh-induced CK1 phosphorylation of Ci directly contributes its optimal transcriptional activity in addition to regulating its stability.

How Hh does signaling differentially regulate these positive and negative phosphorylation events? Previous studies revealed that Ci and its kinases, including PKA, GSK3, and CK1, form protein complexes scaffolded by Cos2 and that Hh signaling induces either dissociation or composition change of these complexes (22, 29, 63), thereby impeding PKA/GSK3/CK1-mediated Ci phosphorylation and proteolytic processing. It is thought that Ci/Gli forms a complex with Sufu in its inactive state and that Hh activates Ci/Gli by dissociating it from Sufu, thus exposing Ci^A/Gli^A to HIB/SPOP and making it vulnerable for ubiquitin/proteasome-mediated degradation (24, 29, 31). It is possible that an Hh-induced change in the formation or composition of Ci/Gli-Sufu complexes makes Ci/Gli more accessible to CK1, allowing CK1-mediated phosphorylation to counteract HIB/SPOP and modulate the speed of Ci/Gli degradation (Fig. 6B). This delicate balance may ensure appropriate levels of Ci^A/Gli^A for Hh signaling, and cells could change this balance to modulate Hh 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 activity can modulate the levels of Ci^A/Gli^A activity in opposite directions raises an interesting possibility that altering CK1 activity may serve as a mechanism for fine-tuning Hh responses in certain contexts.

It has been well established that the Cul1-based E3 ubiquitin ligase SCF complexes recognize substrates upon their phosphorylation, thus linking protein phosphorylation to protein degradation (64). Whether substrate recognition by other Cullin families of E3 ligases also is regulated by phosphorylation remains largely unknown. A previous study showed that replacing the S/T residues in several SPOP degrons with phosphorylated residues blocked binding to SPOP in vitro (46), raising an interesting possibility that recognition of HIB/SPOP substrates could be regulated by kinases. Here we provide the first evidence, to our knowledge, that substrate recognition by Cul3-based E3 ligases is negatively regulated by a kinase. Because Cul3^HIB/SPOP regulates a large family of proteins, our study raises an interesting possibility that the stability of other Cul3^HIB/SPOP targets might also be regulated by phosphorylation.

Materials and Methods

Drosophila Stocks and Transgenes.

The following Drosophila stocks and transgenes were used for this study: CRL, UAS-R*, UAS-Smo^DN (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-CK1ε, and UAS-CK1*(UAS-XCK1ε-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 were inserted 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 Calcium Phosphate Transfection Kit (Specialty Media) according to the manufacturer’s instructions. Hh-conditioned medium was carried out as previously described (17). Sufu^−/− MEFs transfection and the Gli-luciferase reporter assay were carried out as described (66). Immunoprecipitation and Western blot analysis were carried out using standard protocols as previously described (22). The Phos tag-conjugated SDS/PAGE analysis was performed according to standard protocols (48). Phos tag-conjugated acrylamide was purchased from NARD Institute. Immunostaining of imaginal discs was carried out as described (67). Antibodies used for this study were mouse anti-Flag (M2; Sigma), rabbit anti-Flag (Thermo Scientific), mouse anti-HA (F7; Santa Cruz), mouse anti-Myc (9E10; Santa Cruz), phospho-Fu (pT161/pT154) antibody (29), Rat anti-Ci, 2A1 (68), rabbit anti-CKIε (kindly provided by D. M. Virshup, Duke-NUS Graduate Medical School, Singapore), mouse anti-Ptc, and mouse anti-En (Developmental Studies Hybridoma Bank).

RNAi in Drosophila S2 Cells.

DNA templates corresponding to the coding regions of CK1α (nucleotides 601–1014) and CK1ε (nucleotides 834–1323) were generated by PCR and used to make dsRNA targeting the C terminus of CK1α and CK1ε, respectively. dsRNA targeting the Firefly Luciferase coding sequence was used as a control. dsRNAs were generated by MEGAscript High-Yield Transcription Kit (AM1334; Ambion). For the RNAi knockdown experiments, S2 cells first were cultured in serum-free medium containing dsRNA for 12 h at 24 °C. After FBS was added to a final concentration of 10% (vol/vol), dsRNA-treated cells were cultured overnight before transfection with DNA constructs. After additional culturing for 2 d, cells were collected for analysis.

In Vitro Kinase Assay, GST Pull Down, and Protein Stability Assay.

For the in vitro kinase assay, individual GST-fusion proteins bound to glutathione beads were mixed with 0.1 mM ATP containing 10 mCi of γ-³²p-ATP and recombinant CK1 kinase (CK1δ; 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, 10 mM MgCl₂, 1 mM DTT]. Reactions were stopped by adding 4× SDS loading buffer, and the mixture was boiled at 100 °C for 5 min. The phosphorylated GST-fusion proteins were analyzed by autoradiography after SDS/PAGE. The GST pull-down assay was carried out as described (44). The protein stability assay was carried out as described (18).