Sonic Hedgehog Dependent Phosphorylation by CK1α and GRK2 Is Required for Ciliary Accumulation and Activation of Smoothened

Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.


Introduction
The Hh family of secreted proteins plays pivotal roles during embryonic development and adult tissue homeostasis [1][2][3]. Aberrant Hh signaling contributes to numerous human disorders including congenital diseases and cancers [4,5]. In a number of developmental contexts, Hh functions as a morphogen that specifies distinct cell fates in a concentration-dependent manner [1,2]. For example, in vertebrate neural tube patterning, Shh secreted by the notochord and floor pate forms a ventral to dorsal concentration gradient that specifies distinct pools of neural progenitor cells [6].
Hh exerts its biological function through a signaling cascade that ultimately controls a balance between activator and repressor forms of the Gli family of transcription factors [2]. In the absence of Hh, Gli2 and Gli3 are processed into truncated repressor forms (Gli R ). Hh signaling blocks Gli processing and converts full-length Gli2/3 into activator forms (Gli A ). The reception system for the Hh signal consists of a twelve-transmembrane protein Patched (Ptc) as the Hh receptor and a seven-transmembrane protein Smoothened (Smo) as the obligatory Hh signal transducer [2,3]. Ptc inhibits Smo substoichiometrically through a poorly defined mechanism in the absence of Hh [7]. Binding of Hh to Ptc and the Ihog/Cdo family of proteins alleviates Ptc inhibition of Smo [8][9][10][11][12][13][14], leading to Smo activation and signal transduction. How Smo is activated and how it transduces the Hh signal to regulate Gli R and Gli A are still poorly understood.
In mammals, Hh signaling depends on the primary cilium, a microtubule-based membrane protrusion found in almost all mammalian cells [15]. Key components in the Hh pathway are found in cilia and exhibit dynamic patterns depending on the Hh signaling state. For example, in the absence of Hh, Ptc localizes to cilia and prevents Smo from accumulating in the cilia; binding of Hh to Ptc triggers reciprocal trafficking of Ptc and Smo, with Ptc moving out of and Smo accumulating in the cilia [16,17]. Ciliary accumulation of Smo correlates but is not sufficient for Hh pathway activation [16][17][18][19]. Additional mechanisms, including conformational change in Smo, are likely to be responsible for Smo activation [20][21][22]. Indeed, fluorescence resonance energy transfer (FRET) analysis indicates that both Drosophila and mammalian Smo proteins exist as constitutive dimers/oligomers, but in the absence of Hh, Smo C-tails adopt a closed conformation that prevents their association. Hh induces a conformational switch in Smo, leading to dimerization/oligomerization of the Ctails [22]. The mechanisms underlying mammalian Smo ciliary accumulation, conformational change, and activation are largely unknown.
In Drosophila, Hh and Ptc reciprocally control Smo cell surface accumulation and conformation through regulating Smo phosphorylation [22][23][24][25]. In response to Hh, Smo is phosphorylated by protein kinase A (PKA) and casein kinase 1 (CK1) at multiple sites in its C-tail, and these phosphorylation events activate Smo by promoting its cell surface accumulation and active conformation [22,[25][26][27]. However, vertebrate Smo C-tails diverge significantly from that of Drosophila Smo and do not contain the PKA/CK1 phosphorylation clusters found in Drosophila Smo C-tail [22]. In addition, a systematic mutagenesis study did not reveal any Ser/ Thr residues as essential for mammalian Smo activation [28]. These and other observations led to a proposal that mammalian Smo and Drosophila Smo are regulated by fundamentally distinct mechanisms [28,29].
Several studies suggested that G protein coupled receptor kinase 2 (GRK2) positively regulates Shh signaling [30][31][32]. Metabolic labeling experiments revealed that GRK2 is required for the basal phosphorylation of an exogenously expressed Smo [30]. However, it is not clear whether GRK2 directly phosphorylates Smo and how GRK2 activates Shh signaling. In addition, direct evidence for Hh-induced mammalian Smo phosphorylation is lacking. A recent kinome siRNA screen identified CK1a as a positive regulator for Shh signaling, but its mechanism of action remains unknown [33].
In this study, we investigate the activation mechanism of mammalian Smo (henceforth referred to simply as Smo). We demonstrate that Smo is activated via multiple phosphorylation events mediated by CK1a and GRK2 that induce its ciliary accumulation and active conformation. We provide evidence that graded Shh signals induce increasing levels of Smo phosphorylation that fine-tune Smo ciliary localization, conformation, and activity. In addition, we provide evidence that Shh promotes Smo phosphorylation by regulating the accessibility of Smo to its kinases.

CK1a and GRK2 Promote Smo Phosphorylation and Conformational Switch
A previous study revealed that CK1a siRNA blocked Shh pathway activation in C3H10T1/2 cells [33]. To determine how CK1a positively regulates Shh signaling, we tested whether CK1a activates Smo. Coexpression of CK1a with Smo in NIH3T3 cells activated a Gli-luciferase (Gli-luc) reporter gene, although the fold of activation was less dramatic compared with Shh stimulation ( Figure 1A). In line with a previous finding [31], coexpression of GRK2 with Smo also activated Gli-luc in NIH3T3 cells ( Figure 1A). Coexpression of CK1a and GRK2 with Smo had a slightly stronger effect on Gli-luc expression than overexpression of each kinase alone ( Figure 1A). Overexpression of another GRK family member (GRK5) with Smo activated the Gli-luc reporter gene similarly to GRK2 ( Figure 1A), indicating that overexpressed GRK5 and GRK2 have a similar activity in the Shh pathway.
We also examined the effect of CK1a/GRK2 overexpression on Gli-luc expression in the absence of exogenously expressed Smo. Consistent with previous findings [31,33], overexpression of CK1a, GRK2, or both only slightly increased the expression of Gli-luc reporter gene ( Figure S1G). Thus, CK1a/GRK2 overexpression synergized with Smo overexpression to drive Shh pathway activation.
Our previous FRET analysis indicated that Shh induces a conformational change in Smo from a closed to an open conformation [22]. In the closed conformation, Smo exists as a dimer/oligomer through an N-terminal interaction(s), which results in high basal FRET between CFP and YFP fused to the N-termini of two Smo molecules (FRET N ); however, Smo C-tail folds back and is in close proximity to the intracellular loops, resulting in high intramolecular FRET between CFP inserted in the second intracellular loop (L2) and YFP fused to the C-terminus (FRET L2C ) and low intermolecular FRET between CFP and YFP fused to the C-termini of two Smo molecules (FRET C ) (Figure 1B-D) [22]. Shh induced a marked decrease in FRET L2C and a concomitant increase in FRET C without affecting FRET N (Figure 1B-D) [22], suggesting that Smo C-tails move away from the intracellular loops and form dimers/oligomers. To determine whether CK1a and GRK regulate Smo conformation, we carried out FRET analysis using the Smo biosensors indicated in Figure 1B-D. We found that overexpression of CK1a, GRK2, or GRK5 resulted in a significant increase in FRET C ( Figure 1B) and a marked decrease in FRET L2C ( Figure 1C). In contrast, overexpression of these kinases did not cause a significant change in FRET N ( Figure 1D). These results suggest that excessive CK1a and GRK2/5 kinase activities induce a conformational change in Smo similar to that induced by Shh stimulation.
Having established that CK1a and GRK2 act upstream of Smo, we then determined whether CK1a and GRK2 could promote Smo phosphorylation using a Phos-tag gel that specifically retards phosphorylated proteins [34] Here we investigate the molecular mechanism of mammalian Smo (mSmo) activation and find it is similar to that described for Drosophila Smo despite the marked sequence divergence between them. We show that mSmo is activated via phosphorylation at multiple sites by the serine/threonine kinases CK1a and GRK2. We provide evidence that Sonic hedgehog (Shh; the best studied of the three mammalian pathway ligands) can regulate the accessibility of mSmo to these kinases and that phosphorylation promotes the ciliary accumulation of this transmembrane protein in its active conformation. Moreover, increasing concentrations of Shh induce a progressive increase in mSmo phosphorylation that fine-tunes mSmo activity. Thus, our results provide novel insights into the biochemical mechanism of vertebrate Hh signal transduction and reveal a conserved mode of Smo activation.
GRK2. Treating Smo-Myc transfected cells with a Shh-conditioned medium but not a control medium induced a marked mobility shift of Smo-Myc that was abolished by phosphatase treatment ( Figure 1E, lanes 3-4). Importantly, Shh-induced Smo-Myc mobility shift was greatly reduced by treating cells with a CK1 inhibitor CKI-7 [35] and/or a GRK inhibitor heparin [36] ( Figure 1F), suggesting that Shh induces Smo phosphorylation through CK1 and GRK kinase activities.
To establish that CK1a and GRK2 are required for Shhinduced Smo phosphorylation, we generated cell lines stably expressing shRNA targeting CK1a, GRK2, or GRK5. Two independent shRNA constructs that effectively and selectively knocked down the targeted kinase were employed in our assay ( Figure S1A-B). In line with previous findings [31][32][33], CK1a or GRK2 shRNA inhibited Shh pathway activity in the Gli-luc reporter assay ( Figure 1G, Figure S1C-D, H). In contrast, GRK5 shRNA did not alter Shh-induced Gli-luc expression ( Figure 1G, Figure S1E). Importantly, CK1a and/or GRK2 shRNA but not GRK5 shRNA reduced Shh-induced mobility shift of Smo-Myc ( Figure 1H, Figure S1F), suggesting that CK1a and GRK2 are required for Shh-induced Smo phosphorylation. We note that Shh-induced Smo mobility shift was not completely abolished by silencing CK1a and GRK2, likely due to an incomplete elimination of these kinase activities by the RNAi approach ( Figure S1B). However, it is also possible that the residual Smo-Myc phosphorylation in the presence of CK1a and GRK2 shRNA could be due to the involvement of another kinase(s).
For simplicity, we referred to S 592 , S 594 , T 597 , and S 599 collectively as S0; S 615 , S 619 , and S 620 as S1; S 642 as S2; S 666 as S3; S 774 and S 777 as S4; and S 791 as S5 ( Figure 2C). Thus, S1 and S5 are phosphorylation sites for both CK1 and GRK, whereas S0/S4 and S2/S3 are selectively phosphorylated by CK1 and GRK, respectively. Sequence alignment indicates that these phosphorylation sites are conserved among vertebrate Smo proteins ( Figure 2C).
To further characterize Smo phosphorylation in vivo, we attempted to generate phospho-specific antibodies against phosphorylated CK1/GRK sites and succeeded in obtaining an antibody (PS1) that specifically recognizes phosphorylated S1 (pS 615 , pS 619 , and pS 620 , Figure S2F). To monitor phosphorylation at S1, NIH3T3 cells were transfected with Smo-Myc and stimulated with or without Shh-conditioned medium. In the absence of Shh, Smo-Myc exhibited a weak PS1 signal likely due to basal phosphorylation ( Figure 2E, lane 1). Shh induced a clear increase in the intensity of the PS1 signal ( Figure 2E, lane 3). Coexpression of CK1a, GRK2, or both also increased the PS1 signal ( Figure 2E, lanes 5, 7, and 9). On the other hand, Shhstimulated S1 phosphorylation was diminished by treating cells with the CK1 and/or GRK2 kinase inhibitors ( Figure 2E, lanes 11-13). Furthermore, CK1a or GRK2 shRNA reduced whereas combined CK1a/GRK2 shRNA nearly abolished S1 phosphorylation ( Figure 2F, lanes 4, 6, and 8; Figure S2G). In contrast, GRK5 shRNA did not affect S1 phosphorylation ( Figure 2F, lane 10; Figure S2G), consistent with its lack of effect on Shh pathway activity. These results demonstrate that Shh induces S1 phosphorylation by CK1a and GRK2.

Regulation of Smo Phosphorylation by Graded Shh Signals, Oncogenic Mutations, and Small Molecules
Hh signaling strength depends on the level of Hh ligand [2]. To determine if the level of Shh pathway activity correlates with the level of Smo phosphorylation, NIH3T3 cells transfected with Smo-Myc were treated with different levels of Shh, followed by mobility shift assay on the phospho-tag gel or western blot with PS1. We found that increasing levels of Shh induced a progressive increase in the degree of Smo-Myc mobility shift ( Figure 2G), suggesting that Smo-Myc was phosphorylated at more sites in response to higher levels of Shh. In addition, we found that increasing levels of Shh resulted in a gradual increase in the PS1 signal ( Figure 2G), suggesting that the frequency of S1 phosphorylation increases with increasing Shh concentration.
Several oncogenic mutations in human Smo have been identified, including M1 and M2 [39]. The M2 mutation occurs in the seventh transmembrane domain whose murine counterpart is the A1 mutation [20,39]. Previous studies suggest that SmoA1 exhibits constitutive activity, accumulates at primary cilia, and adopts an open conformation [16,20,22]. We found that SmoA1 exhibited slower mobility and elevated PS1 signal intensity regardless of Shh treatment ( Figure 2H, lanes 4-5; Figure S2H) and that A1-induced PS1 signal and mobility shift were abolished by the S1-5 mutation (A1SA1-5) ( Figure 2H, lanes 7-8; Figure  S2H), suggesting that the oncogenic mutation mimics Shh stimulation to induce Smo phosphorylation at CK1/GRK sites. We also observed that M1 increased Smo phosphorylation (see below).

CK1/GRK Phosphorylation Sites Are Essential for Smo Activation
To determine the functional significance of Smo phosphorylation, CK1/GRK sites were mutated to Ala individually or in different combinations (referred to as SA mutation; Figure 2C Figure 3B). Furthermore, the SD variants were further stimulated by Shh, whereas SmoA1 was no longer regulated by Shh ( Figure 3B). Thus, phosphorylation at CK1/GRK sites increases Smo activity in a dose dependent manner but does not confer full activation.
To determine whether the oncogenic mutation activates Smo through its phosphorylation, we mutated several CK1/GRK sites to Ala in SmoA1. Mutating S2/3 (A1SA2, A1SA3, A1SA23) had little if any effect on SmoA1 activity ( Figure 3C). In contrast, S1 mutation (A1SA1) or combined mutations of S1 with other sites (A1SA12, A1SA13, A1SA123, A1SA1-5) greatly reduced or nearly abolished SmoA1 activity ( Figure 3C), suggesting that S1 phosphorylation is critical for the oncogenic mutation to activate Smo. Of note, the SA1 mutation had a more profound effect on the activity of SmoA1 than that of wild type Smo in the presence of Shh (compare SmoA1SA1 with SmoSA1+Shh). The reason for this difference is unclear, but it is possible that the oncogenic mutation may not fully mimic Shh stimulation so that SmoA1 relies on S1 phosphorylation for its activation more than wild type Smo.
We also found that mutating SA0-5 abrogated SAG-induced Smo activation, whereas SD0-5 exhibited resistance to cyclopamine inhibition ( Figure S2I), suggesting that SAG and cyclopamine regulate Smo activity by influencing its phosphorylation.

Mutating CK1/GRK Sites Affects Smo Activity In Vivo
We next used chick neural tubes to determine the role of Smo phosphorylation in Shh signaling in living organisms. CFP-tagged constructs expressing wild type (WT) or mutant forms of Smo were electroporated into one side of the neural tube, leaving the other side as an internal control, followed by immunostaining to visualize the expression of various Hh responsive genes. Electroporation of SmoWT or Smo variants that mimic low-level phosphorylation (SD1, SD12) did not significantly alter the expression of the marker genes ( Figure 3D and Figure S3A); however, electroporation of Smo variants that mimic high-level phosphorylation (SD123, SD1-5, SD0-5) resulted in a dorsal expansion of several ventral markers, including Nkx2.2, Olig2, Nkx6.1, and Islet1 ( Figure 3D and Figure S3D). Furthermore, SD123 and SD0-5 but not SmoWT restored the expression of ventral markers suppressed by a dominant form of Ptc, Ptc1 Dloop2 (PtcD2), as well as prevented the derepression of the dorsal marker Pax7 ( Figure 3E) [42]. These results suggest that phosphorylation at CK1/GRK sites increased the basal activity of Smo in the chick neural tubes.
In line with tissue culture experiments, SmoA1 is more potent than SmoSD variants in inducing ectopic expression of ventral marker genes in chick neural tubes ( Figure 3D, Figure S3B). Mutating S1 (A1SA1) or combination of S1 with other sites to Ala (A1SA12, A1SA13, A1SA123, A1SA1-5) diminished or complete-  Figure S3B), suggesting that phosphorylation at S1 is critical for the oncogenic mutation to activate Smo in the chick neural tube.

Phosphorylation of Smo Promotes Its Ciliary Accumulation
Shh induces ciliary accumulation of Smo that correlates with pathway activation, but the underlying mechanism is poorly understood [16,17]. We determined whether Shh promotes Smo ciliary localization by inducing its phosphorylation at CK1/ GRK sites by examining ciliary localization of CFP-tagged wild type or phosphorylation site mutant forms of Smo in MEF cells treated with or without Shh-conditioned medium. As overexpression by transient transfection caused high basal ciliary localization of Smo, we used retroviral infection to express low levels of exogenous Smo. In these conditions, SmoWT was found in less than 5% of cilia in the absence of Shh but accumulated in ,70% of cilia in response to Shh treatment ( Figure 4A-B). We found that SA mutations inhibited Shh-induced whereas SD mutations promoted basal ciliary accumulation of Smo in a dosedependent manner ( Figure 4A-B). In addition, constitutive ciliary localization of SmoA1 was inhibited by the SA1-5 mutation (A1SA1-5, Figure 4A-B). Thus, phosphorylation at CK1/GRK sites is both necessary and sufficient for the ciliary localization of Smo.
A recent study suggested that b-arrestins mediate Smo ciliary localization by binding to Smo and facilitating its interaction with the kinesin-II motor [43]. We hypothesized that Shh-induced Smo phosphorylation promotes its ciliary localization by recruiting barrestins. To test this possibility, we transfected NIH 3T3 cells with a YFP-tagged b-arrestin2 (b-arr2-YFP) together with Myc-tagged wild type or mutant forms of Smo. As shown in Figure 4, both Shh and the A1 mutation increased the amount of b-arr2 coimmunoprecipitated with Smo ( Figure 4C, lanes 2, 7). The SA mutations nearly abolished Shh-or A1-stimulated interaction ( Figure 4C, lanes 4,10), whereas SD0-5 promoted Smo/b-arr2 interaction ( Figure 4C, lane 5).
We also confirmed that phosphorylation regulates Smo/b-arr2 association using FRET assay. We found that Shh and A1 increased the FRET between Smo-CFP and b-arr2-YFP, and this increase was abolished by the SA0-5 mutation ( Figure 4D  SA0 or SA1 slightly reduced Shh-induced FRET C , whereas individual mutations at other sites (SA2, SA3, SA5) had no effect ( Figure 5A). S0 and S1 double mutation (S01) or combined mutation of S0/1with other sites (SA12, SA13, SA123, SA1-5, SA0-5) greatly reduced or nearly abolished Shh-induced FRET C ( Figure 5A). On the other hand, the SD mutations resulted in a dose-dependent increase in the basal FRET C ( Figure 5B). Overall, the effects of SA or SD mutations on Shh-induced FRET C correlated with their effects on Shh-induced Smo activation.
The SA mutations also diminished A1-induced FRET C ( Figure 5C). Furthermore, SA0-5 abolished SAG-induced FRET C , whereas SD0-5 conferred high basal FRET C even in the presence of cyclopamine ( Figure 5D). Thus, phosphorylation at CK1/GRK sites induced by Shh, A1, and SAG causes a conformational switch in Smo C-tail, leading to its dimerization, whereas cyclopamine locks Smo in the closed conformation by blocking its phosphorylation.

Smo Phosphorylation at the Primary Cilium
To examine the spatial and temporal regulation of Smo phosphorylation, we carried out immunohistochemistry experiments using the PS1 antibody that recognizes phosphorylated S1.  Figure S4A). Cyclopamine induced ciliary accumulation of Smo-CFP but not PS1 ( Figure 6A, Figure  S4A). Furthermore, cyclopamine blocked Shh or 20-OHC but not SAG-induced ciliary PS1 signals ( Figure 6A; Figure S4A (Figure 6B-D). Importantly, we observed a similar kinetics for PS1 accumulation in the primary cilia ( Figure 6B-D). Furthermore, the ratio of PS1 versus Smo-CFP signal intensity in primary cilia remained relatively constant over time. We also monitored Smo phosphorylation in whole cells by western blot using the PS1 and GFP antibodies. We found that the ratio of PS1 versus Smo-CFP signal intensity was lower at early time points and gradually increased over time ( Figure 6E). Thus, Smo phosphorylation exhibited faster kinetics in primary cilia than in whole cells, implying that Smo could be preferentially phosphorylated near or in the primary cilia in response to Shh or SAG, leading to its rapid accumulation in the cilia.

Efficient Smo Phosphorylation Depends on the Kinesin-II Ciliary Motor
To investigate whether primary cilia regulate Smo phosphorylation, we disrupted the cilia using a dominant negative form of Kif3b (DN-Kif3b), a subunit of the kinesin-II motor required for cilia formation [44]. We found that DN-Kif3b diminished but did not completely block Shh-induced PS1 signal associated with either Smo-Myc or SmoA1-Myc ( Figure 6F, lanes 3, 6), suggesting that efficient phosphorylation at S1 depends on the kinesin-II ciliary motor.
We also analyzed whether the primary cilium is required for Shh-induced Smo conformational change by measuring FRET C in the wild type or Kif3a2/2 MEFs transfected with wild type or mutant forms of Smo-CFP C /YFP C . We found that Shh or A1-induced FRET C was dramatically reduced in Kif3a2/2 MEFs compared with WT MEFs ( Figure S4B). In contrast, SmoSD0-5-CFP C /YFP C exhibited high FRET C in both WT and Kif3a2/2 MEFs ( Figure S4B

Shh Promotes Binding of CK1a and GRK2 to Smo
Finally, we investigated how Shh induces Smo phosphorylation by testing the possibility that Shh promotes the accessibility of Smo to its kinases. By immunoprecipitation assay, we found that Shh markedly increased the association between Smo-Myc and endogenous CK1a and GRK2 in NIH3T3 cells ( Figure 7B, lanes 1-2; Figure 7C). In addition, Shh induced accumulation of CK1a in primary cilia ( Figure 7D). The binding of CK1a/GRK2 to  Smo-Myc is specific because we did not detect association between Smo-Myc and endogenous CK1e or GRK5 under the same condition (unpublished data).
To further explore the interactions between Smo and CK1a/ GRK2 and their regulation, we generated several N-or Cterminally truncated forms of Smo ( Figure 7A). As shown in Figure Figure 7G, lanes 3-6). Thus, the CK1a binding pocket is located N-terminal to the phosphorylation sites.
Interestingly, SmoDC588 exhibited increased basal binding to CK1a ( Figure 7G, compare lanes 1 and 5), suggesting that the distal region of Smo C-tail inhibits CK1a binding in the absence of Shh. We hypothesized that unphosphorylated Smo C-tail adopts a closed conformation that could mask the membrane proximal CK1a binding domain ( Figure 7J). Indeed, the SA0-5 mutation, which locked Smo C-tail in its closed conformation, diminished Shh-stimulated CK1a binding, whereas the SD0-5 mutation, which locked the Smo C-tail in its open conformation, increased the basal CK1a binding ( Figure 7B, lanes 3-6; Figure 7C Although kinase binding to Smo is influenced by phosphorylation, we found that Shh still enhanced the binding of CK1a to  Figure 7B, lanes 3-6; Figure 7C). Furthermore, CK1a binding to SmoDC588, which lacks all the CK1/GRK phosphorylate sites, was also upregulated by Shh ( Figure 7G, compare lanes 5 and 6; Figure S5). These results demonstrate that Shh can stimulate CK1a binding through a phosphorylation-independent mechanism. In contrast, GRK2 binding to SmoSD0-5 or SmoSA0-5 was no longer regulated by Shh ( Figure 7B, lanes 3-6), suggesting that Shh promotes GRK2 binding mainly through the phosphorylation-dependent mechanism. Taken together, these data suggest that Shh may regulate CK1a/GRK2 binding in two steps: 1) Shh stimulates CK1a binding to Smo prior to its phosphorylation, which may provide a mechanism to initiate Smo phosphorylation, and 2) phosphorylation of Smo C-tail releases its inhibition on CK1a binding and at the same time increases its binding affinity for GRK2, leading to amplification of Smo phosphorylation ( Figure 7J).

Regulation of CK1a Binding and Smo Phosphorylation by Gain-or Loss-of-Function Smo Mutations
To establish the relationship between kinase association and Smo phosphorylation, we examined how gain-or loss-of-function Smo mutations affect CK1a binding, including two oncogenic mutations (A1 and M1) and three loss-of-function mutations in or near the CK1a binding pocket identified by previous studies ( Figure 7A) [28,39]. We found that both A1 and M1 resulted in a constitutive CK1a/GRK2 binding and Smo phosphorylation with A1 being more potent than M1 ( Figure 7H, lanes 3 and 5; Figure  S5). In addition, Shh further increased the binding of CK1a to and phosphorylation of SmoM1 but not SmoA1 ( Figure 7H, lanes 4 and 6; Figure S5). In contrast, the loss-of-function mutations L430A and S570A blocked Shh-induced CK1a/GRK2 binding and Smo phosphorylation ( Figure 7H, lanes 7-10; Figure S5). Another loss-of-function mutation, I573A, which mainly affected Smo stability [28], slightly reduced Shh-stimulated CK1a binding and Smo phosphorylation ( Figure 7H, lanes 11-12; Figure S5).
If  Figure S5), suggesting that these mutations affect the phosphorylation-independent mechanism that regulates CK1a binding ( Figure 7J). It is possible that the third intracellular loop may also contribute to CK1a binding and this is disrupted by L430A.
In contrast, the M1 and S570 mutations did not affect either the basal or Shh-stimulated binding of CK1a to SmoD588 ( Figure 7G, lanes 9-10 and 13-14; Figure S5). Thus, M1 and S570 affect CK1a binding only in the context of full-length Smo and may act mainly by regulating the release of C-tail inhibition ( Figure 7J).

Discussion
Smo is a central component of the Hh signal transduction cascade and an important cancer drug target, but the molecular mechanism by which Smo is activated has remained poorly understood. In this study, we demonstrate that Smo is activated by multi-site phosphorylation mediated by CK1a and GRK2, and phosphorylation promotes both ciliary localization and active conformation of Smo. We provide evidence that graded Shh signals induce increasing levels of Smo phosphorylation that finetune Smo activity. In addition, we demonstrate that oncogenic mutations and small molecule Hh pathway modulators including SAG, oxysterols, and cyclopamine regulate Smo through CK1a/ GRK2-mediated phosphorylation. We provide evidence that Shh promotes Smo phosphorylation by regulating its accessibility to CK1a/GRK2 and effective Smo phosphorylation depends on the primary cilium. The CK1a/GRK2 sites we identified are conserved among vertebrate Smo proteins; thus, the mechanism we uncover here is likely to be conserved in other vertebrate species.

CK1a and GRK2 Regulate Smo Through Multi-Site Phosphorylation
It has been well established that Drosophila Smo is hyperphosphorylated by multiple kinases in response to Hh stimulation [22,23,[25][26][27]; however, sequence divergence between Drosophila and vertebrate Smo proteins makes it unclear whether vertebrate Smo proteins are similarly phosphorylated in response to Hh. Using the phospho-tag gel and a phospho-specific antibody, we provide the first evidence that Shh induces hyperphosphorylation of Smo, which is mediated by CK1a and GRK2. Several lines of evidence suggest that CK1a and GRK2 are bona fide Smo kinases. First, our in vitro kinase assay with purified Smo fragments and recombinant kinases demonstrated that both CK1 and GRK phosphorylate multiple sites in Smo C-tail. Second, mutating the CK1/GRK sites in the Smo C-tail abolished Shh-stimulated Smo phosphorylation in vivo. Third, using a phospho-specific antibody that recognized an overlapping CK1/ GRK site (S1), we demonstrated that Shh induced phosphorylation at this site through CK1a and GRK2.
We identified a total of six CK1a/GRK2 phosphorylation regions, which we named S0 to S5. S0 and S1 contain multiple phospho-acceptor Ser/Thr residues. Our functional study suggests that S0 and S1 play a major role while other sites play a finetuning role in Smo regulation. The employment of multi-site phosphorylation may allow graded Hh morphogens to induce different levels of Smo activity through differential phosphorylation. Indeed, we found that increasing levels of Shh induced a progressive increase in the level of Smo phosphorylation. Furthermore, increasing the number of SA mutations gradually decreased the level of Shh-induced Smo activity, whereas increasing the number of phospho-mimetic mutations progressively increased the level of basal Smo activity.
Although phospho-mimetic mutations increase the basal activity of Smo both in vitro and in vivo, they do not confer full activation of Smo, which is in contrast to the A1/M2 oncogenic mutation. One possibility is that the SD mutations may not fully mimic phosphorylation and may even lock Smo in a less optimal conformation for activation. However, we think this is unlikely because the SmoSD variants can be further stimulated by Shh to reach their full activity. In addition, phospho-mimetic mutations did not affect SmoA1 activity (unpublished data). These observations suggest that Shh and A1/M2 may stimulate an additional mechanism(s) that acts in conjunction with CK1a/ GRK2-mediated phosphorylation to fully activate Smo. The proposed paralleled mechanisms could be phosphorylationindependent and/or could involve additional kinase(s). Furthermore, although our in vitro and in vivo assays suggest that phosphorylation at S0-S5 is mediated by CK1a/GRK2, we cannot rule out the possibility that some of these sites might also be phosphorylated by other kinases.

A prevalent view regarding Smo activation is that Hh activates
Smo by inducing its ciliary localization [16,17]. However, this view has been challenged by more recent studies showing that the Smo inhibitor cyclopamine promotes instead of blocks ciliary localization of Smo [18,19,45], suggesting that ciliary localization of Smo is insufficient for its activation. Our previous and current studies demonstrate that Shh induces a conformational switch in Smo that is also induced by the A1 mutation and SAG but is blocked by Smo inhibitors including cyclopamine [22,46,47]

Regulation of Smo Phosphorylation
Our data suggested that Shh stimulates Smo phosphorylation, at least in part by regulating the accessibility of Smo to its kinases. Our deletion analyses revealed that CK1a and GRK2 bind Smo through the membrane proximal and distal regions of Smo C-tail, respectively. We provided evidence that Smo C-tail in its closed conformation inhibits CK1a binding likely by masking the membrane proximal CK1a binding pocket through steric hindrance, and this inhibition is released by phosphorylation that promotes the open conformation of Smo C-tail. Furthermore, we demonstrate that Shh stimulates the binding of CK1a to the membrane proximal region of Smo C-tail through a mechanism that parallels with the phosphorylation-dependent mechanism. We propose a two-step mechanism for Shh-regulated kinase association and Smo phosphorylation ( Figure 7J). In the first step (referred to as the initiation step), Shh stimulates CK1a binding to Smo prior to its phosphorylation, likely by inducing a local conformational change near the membrane proximal region that either optimizes the CK1a binding pocket or makes it more accessible to CK1a. This step may contribute to the initiation of Smo phosphorylation and is promoted by the A1 mutation but is blocked by the L430A mutation. In the second step (referred to as the amplification step), CK1a-initiated phosphorylation Our time course study revealed that phosphorylation of Smo occurred more rapidly in the primary cilia compared with the whole cell ( Figure 6). In addition, expression of a dominant negative form of Kif3b, which blocks ciliogenesis, attenuated Shhor A1-induced Smo phosphorylation. These observations suggest that Smo phosphorylation occurs more efficiently in the primary cilia. Interestingly, we found that CK1a is accumulated in primary cilia in response to Shh stimulation ( Figure 7D). The increase in the local concentration of CK1a may explain, at least in part, why phosphorylation of Smo is more effective in the primary cilium. It is also possible that Shh-mediated inhibition of Ptc is more effective in the primary cilium.

Parallels Between Mammalian and Drosophila Smo Activation
Despite the profound difference in the primary sequence between Drosophila and vertebrate Smo, our study suggests that their activation mechanisms are remarkably similar (Figure 8). In both cases, Hh induces Smo phosphorylation at multiple sites (although by distinct sets of kinases) that fine-tune Smo activity, and phosphorylation activates Smo by inducing its active conformation and regulating its subcellular localization (cell surface accumulation for dSmo and ciliary accumulation for mSmo). Hh-stimulated phosphorylation induces dSmo conformation change by antagonizing multiple Arg clusters in its C-tail [22]. As the inactive conformation of mSmo is also maintained by a long stretch of basic cluster in its C-tail [22], multisite phosphorylation may promote mSmo conformational change through a similar mechanism. A recent study has demonstrated that GRK2 regulates dSmo by both kinase-dependent and kinase-independent mechanisms [48]. The observation that Shh induces mSmo/ GRK2 complex formation raises an interesting possibility that GRK2 may also function as a molecular scaffold to promote mSmo activation. cassette. To generate HA-tagged wild type, SA0-5 or SD0-5 versions of Smo C-tail, wild type, or mutant DNA fragments were amplified by PCR and inserted between NotI and XbaI sites in the HA-pUAST vectors [50], and the HA-tagged constructs were subcloned into pCDNA3.1(+) vector with EcoRI and XbaI sites. All the constructs were sequence verified. DN-Kif3b constructs were kindly provided by Dr. Pao-Tien Chuang [44].

Materials and Methods
In Vitro Kinase Assay CK1/GRK in vitro kinase assay was performed according to the manufacturer's instruction (Upstate Biotechnologies, 14-714). Briefly, GST-fusion proteins, 0.1 mM ATP containing 10 mCi of c-32 p-ATP and kinases: CK1d (New England Biolabs), GRK5 (Upstate Biotechnologies, 14-714), were mixed well and incubated at 30uC for 1.5 h in reaction buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 10 mM MgCl2, 1 mM DTT); the reactions were stopped by adding 46SDS loading buffer and boiled at 100uC for 5 min; and the phosphorylation of GST-fusion proteins were analyzed by autoradiography after SDS-PAGE.

Cell Cultures
Unless otherwise noted, all the mammalian cell lines were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), L-glutamine, 1 mM sodium pyruvate, and penicillin. NIH 3T3 cells were obtained from ATCC. smo 2/2 and Kif3a 2/2 mouse embryonic fibroblasts were kindly provided by Dr. Pao-Tien Chuang [44]. Wild type MEFs were derived from wild type mice embryos at 9.5 dpc, embryos were dissected to pieces and transferred to 10 cm dishes for adherence, regular DMEM medium were slowly added, fibroblasts cells that migrated from the embryos were collected by trypsinization after 3,5 d, and expanded wild-type MEFs were aliquot and frozen for further use. Reagents were used in the following concentrations unless otherwise noted: Recombinant Mouse Sonic Hedgehog Nterminus (ShhNp, R&D systems, Cat #464-SH), 293-Shhconditioned medium (1:6 v/v; [40]), SAG (200 nM), cyclopamine (1 mM), CKI-7 (10 mM; Sigma), and Heparin (1 mM; Sigma). SAG and cyclopamine are gifts from Dr. James Chen at Stanford University. The kinase inhibitors were added into the medium the night before collecting the samples, and for heparin treatment, 5 mg/ml Lipofectin (Invitrogen) were mixed together with the medium to facilitate their entry into the cells.
Transfection, Immunoprecipitation, Western Blot, Immunochemistry, and FRET For protein expression, cells were transfected with FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, harvested and lysed in RIPA buffer (50 mM Tris-Cl at pH 7.9, 150 mM NaCl, 5 mM EDTA), 1% NP-40 supplemented with protease inhibitors (Roche), and lysates were frozen and thaw 2,3 times. Immunoprecipitation experiments were performed as previously described [51]. The Phos tag-conjugated SDS-PAGE analysis was performed according to the standard protocols [34]. Phos tag-conjugated acrylamide was purchased from the NARD Institute in Japan. First and secondary antibodies used in this study: mouse anti-Myc (1:5,000; Sigma), rabbit anti-Myc (A-14; Santa Cruz Biotechnologies), mouse anti-HA (1:10,000; Santa Cruz Biotechnologies), mouse anti-Flag (1:10,000; Santa Cruz Biotechnologies), rabbit anti-CK1a (Santa Cruz Biotechnologies), rabbit anti-GRK2 (Santa Cruz Biotechnologies), rabbit anti-GRK5 (Santa Cruz Biotechnologies), rabbit phospho-specific antibodies against S1 (PS1, 1:50), monoclonal anti-Acetylated tubulin (1:1,000; Sigam#T7451), Goat anti-mouse IgG HRP (1:10,000), and Goat anti-rabbit IgG HRP (1:10,000). PS1 antibody was generated by Genemed Synthesis Inc., phosphorylated peptide EP(pS)ADV(pSpS)AWAQHVTC was injected into rabbit, the serum was affinity-purified by antigen, and the flow-through from the affinity-purification was also kept as control antibody S1 against non-phosphorylated peptide. For immunofluorescence, cells were seeded on ploy-D Lysine coated LAB-TEK chamber slides and were transfected with indicated constructs, followed by treating with indicated reagents for indicated time. Cells were washed 2 times with 1XPBS and fixed with 4% PFA, permeabilized, stained, and mounted for observation with Zeiss LSM510 confocal microscope. FRET assays were performed essentially as previously described [22]. Briefly, CFP was exited at 458 nM wavelength and YFP at 514 nM wavelength. CFP signals were collected once before photobleaching (BP) and once after photobleaching (AP) of YFP. YFP was photobleached with full power of the 514 nM laser line for 1,2 min at the top half of the cells, leaving the bottom half as an internal control. The CFP signals from the bleached half (both membrane and cytoplasmic signals) were used for FRET calculating, and the efficiency of FRET was calculated with the formula: FRET% = [(CFP AP 2 CFP BP )/CFP AP ] 6100.

8XGliBS-Luciferase Assay
The day before transfection, different cell lines were seeded at a density of 1,2610 5 cells/ml in 24-well plates, and cells were transfected with 8XGliBS-luciferase and pRL-TK at 4:1 ratio, and 5% w/w of pGE-Smo constructs with Fugene 6 (Roche) according to the manufacturer's instructions. After 2 d of transfection, cells were changed to low serum medium (DMEM supplemented with 0.5% calf serum) with or without Shh-conditioned medium combined with additional treatments as indicated, and cells were harvested and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) and FLUOstar OPTIMA (BMGLABTCH). Each sample was performed in triplicate and the assays were repeated for at least 3 times.

Retroviral Infection and shRNA
Stable NIH 3T3/shRNA cell lines against kinase CK1a, GRK2, or GRK5 were generated by retroviral infection and selected with 3 mg/ml of puromycin.
HEK 293T cells were transfected with XZ201 retrovirus vectors encoding variant Smo cDNAs and pCL-Eco packaging vector, and supernatants were collected 72 h post-transfection, filtered through a 0.45 mM syringe filter, and added to 50,70% confluent wild type MEFs with 8 mg/ml polybrene (Sigma) overnight.  Figure 7G and Figure 7H. The pull-downed CK1a signal intensity in each lane was normalized by the pull-downed Smo signal intensity and compared with lane 1. *p,0.05, **p,0.01, ***p,0.005. The signal intensity for each band was quantified by ImageJ software followed by Prism analysis, n = 3. (TIF)