Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase

Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.


Introduction
Hedgehog (Hh) signaling plays an essential role in embryonic development and adult tissue homeostasis, and its deregulation has been implicated in congenital diseases and cancers [1][2][3][4][5][6]. Hh exerts its biological influence through an intracellular signal transduction cascade that emanates from a G protein coupled receptor (GPCR)-family protein Smoothened (Smo) and culminates in the activation of the latent transcription factor Cubitus interruptus (Ci)/Gliomaassociated oncogene homologue (Gli) [1,3,7,8]. In the signaling off state, Smo is inhibited by a twelve-transmembrane protein Patched (Ptc). Binding of Hh to Ptc alleviates such inhibition, allowing Smo to be phosphorylated and accumulate on the cell surface in Drosophila or primary cilia in vertebrates, where Smo adopts an open and active conformation to relay the Hh signal to the intracellular signaling components [9][10][11][12][13][14][15].
Phosphorylation plays a critical role in the regulation of Smo conformation and subcellular localization [16]. In Drosophila, unphosphorylated or hypophosphorylated Smo adopts a closed, inactive conformation [12] and is ubiquitinated and removed from the cell surface by both proteasome-and lysosome-mediated degradation [17,18]. Upon Hh stimulation, Smo is phosphorylated by protein kinase A (PKA) and casein kinase 1 (CK1), mainly the CK1α/ε isoforms, at three clusters of Ser/Thr residues in its carboxyl-terminal intracellular tail (C-tail) [19][20][21][22], which drives a conformational switch of Smo C-tail from the closed inactive to an open active conformation, leading to dimerization/oligomerization of the C-tail [12]. In addition, PKA/CK1α/ε-mediated phosphorylation inhibits ubiquitination of Smo, leading to its cell surface accumulation and activation [17,18]. In addition to PKA and CK1α/ε, Smo activity is also modulated by casein kinase 2 (CK2), atypical protein kinase C (aPKC), and G protein coupled receptor kinase 2 (Gprk2) [23][24][25][26]. Gprk2 promotes Smo activity by directly binding and phosphorylating the Smo C-tail to stabilize its active conformation [24]. In mammals, Hhstimulated phosphorylation of Smo by CK1α and Gprk2 promotes its ciliary localization and active conformation [15]. Cell surface/ciliary accumulation of Smo is thought to be essential for its activation and function [14,27]; however, the underlying mechanism is still poorly understood.
Hh functions as a morphogen to specify different developmental outcomes in a concentration-dependent manner [1,3]. As such, Hh signal transduction needs to be tightly controlled to achieve pathway activities appropriate with the ligand inputs. Although the characterized phosphorylation events contribute to Smo activation, they do not represent all the activation mechanisms, as phospho-mimetic mutations failed to fully activate Smo in both Drosophila and mammals [15,19,24], suggesting that additional mechanisms, either phosphorylationdependent or independent, may exist. Indeed, Drosophila Smo is phosphorylated at more than 26 Serine (Ser)/Threonine (Thr) residues, many of which have not been well characterized [20].
CK1γ, which is encoded by gilgamesh (gish) in Drosophila [28], is a membrane-associated Ser/Thr kinase of the CK1 family [29]. Gish/CK1γ has been implicated in the regulation of Wingless (Wg)/Wnt signaling by phosphorylating the co-receptor Arrow (Arr)/LRP5/6 [30,31]. Gish is also involved in glial cell migration in Drosophila eye [28], olfactory learning [32], and planar cell polarity (PCP)-mediated morphogenesis [33]. Mammals have three CK1γ isoforms encoded by different genes [29], making it difficult to study the role of CK1γ in development. In this study, we identified Gish as a positive regulator of Hh signaling through a genetic modifier screen. We demonstrated that Hh stimulates the association between Gish and Smo in a manner depending on cell surface localization of both Gish and Smo as well as PKA-mediated phosphorylation of Smo C-tail. We provided evidence that Gish phosphorylates a membrane proximal region of Smo C-tail to promote high levels of Hh pathway activity. However, loss of Gish only caused a minor defect in Hh pathway activity, likely due to a redundancy with another kinase(s). We also found that vertebrate CK1γ is localized at primary cilia depending on its membrane association, and that CK1γ promotes Smo phosphorylation and Sonic hedgehog (Shh) pathway activation depending on the primary cilia. Our results suggest that plasma membrane/ciliary-localized CK1γ plays a conserved role in Hh signaling by promoting the maximal levels of Smo activity.

A Genetic Modifier Screen Identified Gish as a Positive Regulator of the Hh Pathway
To identify additional Hh pathway regulators, we have conducted an RNAi-based genetic modifier screen to identify enhancers or suppressors of a "fused wing" phenotype caused by expression of a dominant negative Smo (Smo -PKA12 /Smo DN ) with a wing-specific Gal4 drivers MS1096 (MS>Smo DN ) [19,24,25]. We found that expressing a UAS-RNAi line (V106826) targeting Gish, the Drosophila homologue of mammalian CK1γ, enhanced the "fused wing" phenotype caused by MS>Smo DN (Fig 1A-1C). Using immunostaining with a Gish antibody, we found that Gish RNAi effectively knocked down Gish protein expression in wing imaginal discs (S1A-S1B' Fig). Moreover, two additional UAS-Gish RNAi lines, V26003 and BL28066, enhanced the MS>Smo DN -induced phenotype in a similar fashion (S1C-S1F Fig). On the other hand, overexpression of a wild-type Gish, but not a kinase dead form (Gish KD ), partially suppressed the "fused wing" phenotype caused by MS>Smo DN (Fig 1D and 1E and S1K and S1L Fig).
Gish/CK1γ is localized to the plasma membrane due to its C-terminal palmitoylation [30,33]. Interestingly, overexpression of a soluble form of Gish with its palmitoylation site deleted, GishΔC [33], failed to rescue MS>Smo DN -induced wing phenotype, even though it was expressed at levels similar to the wild-type Gish (Fig 1F and S1M Fig compared with S1K  Fig), suggesting that plasma membrane association of Gish is critical for its function in this context. Consistent with this notion, overexpression of other soluble CK1 family members, including CK1α and CK1ε, also failed to rescue the MS>SmoDN phenotype (Fig 1G and 1H).
To determine whether loss-or gain-of-Gish function modified the "fused wing" phenotype through the Hh pathway, we examined the expression of an Hh target gene ptc. In control late third instar wing imaginal discs, Hh induced ptc expression in A-compartment cells near the A/P boundary (Fig 1A'). In MS>Smo DN wing discs, ptc expression near the A/P boundary was greatly reduced (Fig 1B'). Gish RNAi in MS>Smo DN wing discs nearly abolished ptc expression near the A/P boundary in the wing pouch region where MS1096 was expressed (Fig 1C'). On the other hand, overexpression of Gish restored ptc expression close to wild-type levels ( Fig  1D'). Hence, gain-or loss-of-Gish activity can modulate Hh pathway activity.
To determine where Gish acts in the Hh pathway, we examined its genetic interaction with Fused (Fu), a Ser/Thr kinase acting downstream of Smo [11,34,35]. Inactivation of Fu by expressing a USA-RNAi transgene with MS1096 (MS>Fu RNAi ) caused a similar "fused wing" phenotype, albeit more severe than that caused by MS>Smo DN (S1H Fig). However, neither Gish overexpression nor RNAi modified the wing phenotype caused by MS>Fu RNAi (S1I and S1J Fig), suggesting that Gish may act upstream of Fu in the Hh pathway.

Characterization of gish Mutants
To confirm the Gish RNAi phenotype, we turned to gish mutants. gish KG03891 is a P-element insertion mutation and a strong allele of gish [32], which is referred to as gish P hereafter. Although gish P heterozygosity did not modify the fused wing phenotype caused by MS>Smo DN (Fig 2B compared with Fig 2A), MS>Smo DN wings carrying gish P homozygous clones exhibited a greatly enhanced phenotype similar to that caused by Gish RNAi in MS>Smo DN wings ( Fig  2C compared with Fig 1C and S1E and S1F Fig). Interestingly, we found that heterozygosity for gish deficiency, Df(3R)ED10639 (BL#9481), also enhanced the fused wing phenotype caused by MS>Smo DN (Fig 2D), suggesting that gish P is not a null allele. To obtain a gish null allele, we generated imprecise excision lines from gish P and found several lines including gish Δ4 that could enhance the MS>Smo DN phenotype similarly to gish Df (Fig 2E). Consistent with gish Δ4 being a null allele, gish Δ4 mutant clones in wing discs exhibited diminished Gish immunostaining (S2A and S2B Fig). Furthermore, the enhancement of the MS>Smo DN phenotype by gish Δ4/+ was reversed by coexpression of the wild-type Gish but not by coexpression of either Gish KD or Gish ΔC (Fig 2F-2H).
Our previous study identified Gprk2 as a positive regulator of Hh signaling in a similar genetic modifier screen [24]. Heterozygosity of a gprk2 null allele, gprk2 Δ15 , also enhanced the fused wing phenotype caused by MS>Smo DN (Fig 2I). Interestingly, taking away one copy of gish in this background (MS>Smo DN gish Δ4/+ gprk2 Δ15/+) further enhanced the wing phenotype ( Fig 2J). This dosage-sensitive genetic interaction between Gish and Gprk2 suggests that they may act in close proximity in the Hh signaling pathway.

Gish Is Required for High-Level Hh Pathway Activity
We then induced gish Δ4 clones using the MARCM system [36] at 24-48 h or 48-72 h after egg laying (AEL). gish Δ4 clones (marked by green fluorescent protein [GFP] expression) induced at 48-72 h AEL survived to late third instar larval stages but did not affect the expression of Hh target genes ptc and engrailed (en) when localized in A-compartment cells near the A/P boundary (S2D-S2D " Fig and Fig 2F-2F" compared with S2C-S2C" and S2E-S2E" Fig). gish Δ4 clones induced at 24-48 h AEL were barely recovered, suggesting that gish mutant cells had a growth disadvantage and were competed out by wild-type cells over time. To recover early-induced clones, we generated gish Δ4 clones in a Minute background, which gave gish mutant cells a growth advantage [37]. As shown in Fig 2 and S2 Fig, gish Δ4 clones (marked by the lack of Gish staining) induced at 24-48 h AEL in the Minute background occupied large areas in late third instar wing discs ( Fig 2L and 2N, S2H Fig). ptc expression, which is induced by intermediate levels of Hh, was not affected in gish Δ4 clones (S2H" and S2H‴ Fig); however, en expression in A-compartment cells near the A/P boundary, which is induced by peak levels of Hh, was diminished in gish Δ4 clones (arrow in Fig 2L-2L‴). In addition, Hh-induced Smo accumulation appeared to be attenuated in gish Δ4 clones (arrow in Fig 2N-2N‴). These results suggest that Gish is required for high levels of Hh signaling activity and may regulate the Hh pathway at the level of Smo.

Gish Phosphorylates the Membrane Proximal Region of Smo C-tail
A previous study revealed that Smo derived from Hh-stimulated S2 cells was phosphorylated at 26 Ser/Thr residues in its C-tail, including the three PKA/CK1 phosphorylation clusters and Gprk2 sites (Fig 3A) [20]. However, the kinases responsible for phosphorylating other sites, most notably, a membrane proximal cluster (CL-II) of Ser/Thr residues, 623 DlNSSETNDISS 634 (underlined S/T residues were phosphorylated sites detected by Mass Spec) [20], have not been identified ( Fig 3A). A close inspection of the CL-II site indicates that it contains Ser/Thr residues falling into the consensus sites for the CK1 family kinases: D/E/(p)S/T(X) 1-3 S/T, in which the underlined S/T is the CK1 site, whereas X represents any amino acid [29]. Indeed, a Glutathione S-transferase (GST) fusion protein containing the intact (GST-Smo 601-700 ) but not the mutated CL-II site (GST-Smo 601-700 CL-II SA) was phosphorylated by a recombinant CK1 in an in vitro kinase assay ( Fig 3B). To determine whether CL-II is phosphorylated by Gish, we transfected S2 cells with a Smo construct, Myc-Smo4C650, which contains the CL-II site but lacks distal phosphorylation sites such as the PKA/CK1 phosphorylation clusters and Gprk2 sites. We found that Hh stimulation induced a mobility shift of Myc-Smo4C650, which is indicative of Smo phosphorylation [19], and that Hh-induced mobility shift of Myc-Smo4C650 was diminished by Gish RNAi (Fig 3C). Coexpression of Myc-Smo4C650 with Gish also induced a mobility shift of Myc-Smo4C650 (Fig 3C and 3D). Furthermore, mutating the CL-II site in Myc-Smo4C650 (Myc-Smo4C650CL-IISA) abolished its mobility shift induced by either Hh stimulation or Gish overexpression ( Fig 3D). Taken together, these results suggest that Hh induces phosphorylation of CL-II through Gish.

Hh Stimulates CL-II Phosphorylation by Plasma Membrane-Associated Gish
To characterize CL-II phosphorylation in the context of full-length Smo, we generated a phospho-specific antibody named Smo4P using the phospho-peptide NDLN( P S)( P S)E( P T)NDI( P S)  Gprk2 is responsible for CL-II phosphorylation [38]; however, we found that Gprk2 RNAi or overexpression did not affect Smo4P signal intensity associated with Myc-Smo (S3A and S3B  Fig). Furthermore, treatment of Myc-Smo-expressing cells with a pharmacological CK1 inhibitor, D4476, also abolished Hh-stimulated Smo4P signal (Fig 3G and 3H). These results suggest that Hh stimulates CL-II phosphorylation through Gish rather than Gprk2.
Gish/CK1γ is attached to the inner leaf of the plasma membrane through its C-terminal palmitoylation ( Fig 4A) [30]. Indeed, HA-Gish was mainly associated with cell membrane when expressed in S2 cells (Fig 4B). By contrast, HA-Gish CS and HA-Gish ΔC , which have their C-terminal palmitoylation signal (SRCCCFFKR) substituted (SRSSSFFKR) or deleted, respectively (Fig 4A), exhibited cytoplasmic distribution ( Fig 4B) [33]. We then coexpressed HA-Gish, HA-Gish CS , or HA-Gish ΔC with Myc-Smo in S2 cells with endogenous Gish knocked down by dsRNA targeting the 5' UTR of gish. Western blot analysis with Smo4P indicated that only the membrane-associated form of Gish (HA-Gish) but not the cytosolic variants (HA-Gish CS and HA-Gish ΔC ) could support CL-II phosphorylation in response to Hh (Fig 4C), suggesting that plasma membrane association of Gish is critical for CL-II phosphorylation. In addition, we found that coexpression of the cytosolic CK1 family members CK1α and CK1ε with Myc-Smo did not significantly increase the Smo4P signal in either the presence or absence of Hh stimulation ( Fig 4D and S3C Fig). These results may explain why overexpression of wild-type Gish but not Gish ΔC , CK1α, or CK1ε could partially rescue the wing phenotype caused by MS-Smo DN (Fig 1D and 1F-1H).

CL-II Phosphorylation Depends on Cell Surface Localization of Smo
To determine whether cell surface localization of Smo is important for CL-II phosphorylation, we employed a Myc-tagged Smo variant that has a ubiquitin (Ub) moiety fused to its C-terminus (Myc-Smo-Ub) [18]. We confirmed the previous finding that Myc-Smo-Ub failed to accumulate on the cell surface in response to Hh ( Fig 4E) [18,39]

CL-II Phosphorylation Depends on PKA-Mediated Phosphorylation of Smo
Previous studies suggest that Hh stimulates Smo phosphorylation by PKA and CK1α/ε at three clusters of Ser/Thr residues in the middle region of Smo C-tail (Fig 3A), and that these phosphorylation events promote Smo cell surface accumulation and conformational change [12,19]. To determine whether PKA-mediated phosphorylation of the distal sites regulates Gish-mediated phosphorylation of the membrane proximal sites, we first

Differential Regulation of CL-II and PKA Site Phosphorylation by Hh
We next determined whether PKA site phosphorylation and CL-II phosphorylation were induced by different levels of Hh. S2 cells stably expressing Myc-Smo were treated with Hhconditioned medium containing different levels of Hh-N (20%, 40%, 60%, 80%, or 100%) for 4 h. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with either anti-SmoP687, which recognized the phosphorylated PKA site (S687) as well as two downstream CK1 sites [40], or anti-Smo4P. As shown in Fig 4I, Fig 4J). Hence, the CL-II phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than the PKA-site phosphorylation.

CL-II Phosphorylation Is Required for Optimal Smo Activity
To determine the functional importance of CL-II phosphorylation, we mutated CL-II in the context of Smo-cyan fluorescent protein (CFP) or SmoSD123-CFP to generate SmoCL-II-SA-CFP and SmoSDCL-IISA-CFP, respectively (see Materials and Methods). Consistent with previous findings [11,19], overexpression of Smo-CFP using the MS1096 Gal4 driver (MS>Smo-CFP) induced ectopic expression of dpp-lacZ, which is a low-threshold Hh target gene ( Fig 5A); however, SmoCL-IISA-CFP failed to induce ectopic dpp-lacZ expression ( Fig  5B). While MS>SmoSD123-CFP induced ectopic expression of not only dpp-lacZ but also ptc-lacZ and en at high levels ( Fig 5C-5C"), SmoSDCL-IISA-CFP failed to induce ectopic expression of en and only induced ectopic expression of ptc-lacZ at low levels, although the ectopic expression of dpp-lacZ was not affected by the CL-IISA mutation (Fig 5D-5D To discern the in vivo function of CL-II phosphorylation more precisely, we expressed Smo-CFP and SmoCL-IISA-CFP at low levels using the weak Gal4 driver C765 (C765>Smo) in wing discs carrying smo 3 mutant clones. Our previous study revealed that the levels of Smo derived from C765>Smo were only slightly higher than that of endogenous Smo [22]. We found that C765>Smo-CFP completely rescued the expression of both ptc and en in smo 3 mutant clones located near the A/P boundary (Fig 5G-5H" compared with Fig 5E-5F"). By contrast, C765>SmoCL-IISA-CFP failed to rescue en expression and only partially rescued ptc expression in smo 3 clones (Fig 5I-5J"). In addition, A/P boundary-located smo 3 cells expressing C765>SmoCL-IISA-CFP accumulated high levels of full-length Ci (Fig 5I'), suggesting Smo-CL-IISA-CFP could still inhibit Ci processing but fail to induce the maturation of full-length Ci into the active but labile form [41]. Furthermore, we found that SmoCL-IISA-CFP, like Smo-CFP, promoted Ci nuclear localization in smo mutant clones (S5E-S5F 000 Fig). Previous studies revealed that Ci nuclear translocation occurs in A-compartment cells more than ten cells away from the A/P boundary, where there are low levels of Hh [42,43]. Collectively, these results demonstrate that CL-II phosphorylation is essential for Smo to transduce high levels of Hh signaling activity but is dispensable for low levels of Hh pathway activity.

Gish-and Gprk2-Mediated Phosphorylation Promotes High Levels of Smo Activity
Our previous study demonstrated that Gprk2 promotes high levels of Hh pathway activity by regulating the active state of Smo through both kinase activity dependent and independent mechanisms [24]. Gprk2 phosphorylated Smo at Ser741/Thr742 and S1013/S1015, and mutating these sites (GPSA12) in the context of SmoSD123 (SmoSDGPSA) compromised Hh SmoSD123 induced ectopic ptc-lacZ expression in A-compartment cells both distant from and close to the A/P boundary, albeit at levels lower than that of endogenous ptc-lacZ at the A/P boundary (Fig 6A and 6A'). In addition, SmoSD123 induced weak ectopic en expression in Acompartment cells near the A/P boundary (Fig 6A") (Fig 6C-6D"). Although both SmoSDall and SmoSDGPSD induced ectopic ptc-lacZ expression at similar levels ( Fig 6C' and 6D'), SmoSDall induced ectopic en expression in more anterior cells than SmoSDGPSD (Fig 6C" and 6D"). We also examined the activity of several Smo variants with different phospho-mimetic mutations in Clone 8 (Cl8) cells through a ptc-luciferase reporter assay. As shown in Fig 6E,  Our previous studies revealed that phosphorylation of Smo C-tail induced a conformational change resulting in its dimerization/oligomerization, as indicated by an increased fluorescence resonance energy transfer (FRET) between C-terminally tagged CFP and yellow fluorescent protein (YFP) [12,24]. We found that mutating the CL-II site in SmoSD123 reduced its C-terminal FRET (Fig 6F), suggesting that CL-II phosphorylation may promote the active Smo conformation.  (Fig 7E), suggesting that Smo interacts with Gish through its C-tail. HA-Gish was also associated with Myc-SmoΔC650 in S2 cells ( Fig 7E). Furthermore, this association was enhanced upon Hh stimulation (Fig 7F), suggesting that Hh signaling facilitates the binding of Gish to the membrane proximal region of the Smo C-tail.

Smo and Gish Form a Complex Stimulated by Hh
Finally, we determined whether Smo-Gish association is regulated by PKA-mediated phosphorylation of Smo. We found that mutating the PKA phosphorylation sites to Ala (SA123) attenuated Hh-stimulated Smo-Gish complex formation, whereas the phospho-mimetic mutations of PKA sites and adjacent CK1 sites (SD123) increased both the basal and Hh-stimulated Smo-Gish complex (Fig 7G). The influence of PKA phosphorylation of Smo on Smo-Gish interaction could explain why CL-II phosphorylation is affected by PKA (Fig 4G and 4H).

Ciliary Localized CK1γ Regulates Smo Phosphorylation and Shh Signaling
We next determine whether CK1γ regulates Shh signaling in mammalian cells. When expressed in NIH3T3 cells, an enhanced yellow fluorescent protein (EYFP)-tagged Xenopus CK1γ (EYFP-CK1γ) was accumulated on the plasma membrane as well as on the primary cilium, whereas a cytosolic form of CK1γ, EYFP-CK1γ-ΔC, which lacks the C-terminal palmitoylation site [30], failed to localize on the primary cilium (Fig 8A), suggesting that ciliary localization of CK1γ depends on its plasma-membrane association. In a Gli-luciferase (Gli-luc) reporter assay, EYFP-CK1γ but not EYFP-CK1γ-ΔC stimulated Shh pathway activity (Fig 8B). By contrast, two dominant-negative forms of CK1γ CK1γ K73R and CK1γ D164N , which specifically inhibited CK1γ activity [30], suppressed Shh-stimulated Gli-luc reporter activity (Fig 8C). Our previous study revealed that Shh stimulated the phosphorylation of mammalian Smo Ctail at multiple clusters of Ser/Thr residues, including a membrane-proximal cluster (S1) important for Smo activation [15]. Western blot analysis using an antibody (PS1), which recognizes phosphorylated S1 site [15], indicated that overexpression of EYFP-CK1γ but not EYFP-CK1γ-ΔC could stimulate Smo phosphorylation at the S1 site (Fig 8E). On the other hand, overexpression of either CK1γ K73R or CK1γ D164N attenuated Shh-stimulated S1 phosphorylation (Fig 8F). We found that EYFP-CK1γ and Myc-Smo formed a complex when coexpressed in NIH3T3 cells and that CK1γ/Smo association was enhanced upon Shh stimulation (Fig 8H). By contrast, EYFP-CK1γ-ΔC failed to interact with Smo even in the presence of Shh (Fig 8H). Because EYFP-CK1γ-ΔC failed to accumulate on the primary cilium, we speculated that the function of CK1γ in the regulation of Smo phosphorylation and Shh pathway activity might depend on the primary cilium. To disrupt the primary cilium, we overexpressed a dominant negative form of Kif3b (DN-Kif3b), a subunit of the Kinesin-II motor required for the cilium formation [15,44]. Indeed, DN-Kif3b overexpression blocked CK1γYFP/Myc-Smo association (Fig 8I), CK1γ-stimulated S1 phosphorylation (Fig 8G), and Gli-luc reporter activity (Fig 8D), suggesting that the primary cilium is required for CK1γ to bind and phosphorylate Smo. A recent study revealed that the cell-membrane-permeable Smo agonist SAG could activate Smo without its ciliary accumulation [45]. Indeed, we found that SAG stimulated Smo phosphorylation at the S1 site in NIH3T3 cells transfected with DN-Kif3b, although Shh failed to  stimulate Smo phosphorylation in these cells (Fig 8J), suggesting that SAG can induce Smo phosphorylation in the absence of the primary cilia, albeit at lower efficiency than in the presence of the primary cilia. This cilium-independent Smo phosphorylation was abolished when cells were transfected with a dominant negative form of CK1α (CK1α DN [46]); however, the dominant negative forms of CK1γ (CK1γ K73R and CK1γ D164N ) failed to block cilium-independent Smo phosphorylation induced by SAG (Fig 8J). These results further support the notion that CK1γ regulates Smo phosphorylation in the primary cilium.

Discussion
In this study, we identified a plasma membrane-associated kinase Gish/CK1γ as a conserved positive regulator of the Hh pathway. We found that Hh stimulated the binding of Gish/CK1γ to Smo to phosphorylate a Ser/Thr cluster located in the membrane-proximal region of the Smo C-tail (CL-II site in Drosophila Smo and S1 site in mammalian Smo). We demonstrated that CL-II phosphorylation is required for the optimal Smo activation and Hh pathway activity. Interestingly, we found that Gish/CK1γ regulates Smo phosphorylation and Hh pathway activity depending on its plasma membrane/ciliary localization. We propose that cell surface/ciliary accumulation of Smo, which is facilitated by Hh stimulation, promotes its association, phosphorylation, and activation by Gish/CK1γ (Fig 9).
As an obligatory transducer of the Hh signal, Smo relays the positional information imposed by Hh morphogen gradient to different levels of pathway activity that elicit distinct developmental outcomes. How Smo is differentially activated by Hh morphogen gradient is incompletely understood; however, it has been suggested that different levels of Hh induce different levels of Smo phosphorylation, which are in turn translated into different levels of Hh pathway activity [16]. Indeed, it has been observed that increasing levels of Hh resulted in a progressive increase in the overall levels of Smo phosphorylation, and that increasing the number of phospho-mimetic mutation in the Smo C-tail led to a gradual increase in Hh pathway activity [11,12,15,40]. A previous study revealed that, in cultured Drosophila cells exposed to Hh, Smo was phosphorylated on at least 26 Ser/Thr residues in its C-tail [20]. While the distal phosphorylation sites including the three PKA/CK1 phosphorylation clusters have been well characterized, the biological function of and the kinases involved in the phosphorylation of the membrane-proximal sites, most notably the CL-II site, have remained largely unexplored. In addition, it remains unclear whether phosphorylation of CL-II is constitutive or stimulated by Hh because its phosphorylation state was not determined in the absence of Hh stimulation.
In this study, we generated an antibody (Smo4P) that recognized phosphorylated CL-II and demonstrated that CL-II phosphorylation was stimulated by Hh. Moreover, we found that CL-II phosphorylation was mediated by Gish in a manner depending on its membrane association. Both gish mutation and phosphorylation deficient CL-II mutation affected the expression of only high-threshold Hh target genes such as en, suggesting that Gish-mediated CL-II phosphorylation is not absolutely required for Hh signal transduction but rather plays a modulatory function in fine-tuning Hh signaling strength. We noticed that the reduction of Hh pathway activity in gish mutant clones was less severe compared with that associated with CL-II mutation (Fig 2, S2 Fig and Fig 5). One possibility is that we did not completely overcome the perdurance issue even when gish clones were generated at 24-48 h AEL. The early-expressed gish gene products could persist even after many rounds of cell division, and residual Gish kinase activity might still exist in gish mutant cells to partially phosphorylate CL-II. Indeed, we didn't observe any change in Hh pathway target gene expression in gish mutant clones generated at 48-72 h AEL, many of which are >30 cells in size, suggesting that gish gene products generated at early time points survived many rounds of cell divisions. Another possibility is that in the absence of Gish or when Gish activity is compromised, another kinase(s)-for example, other members of CK1 family-may partially compensate for the loss of Gish, although these kinases do not phosphorylate CL-II as effectively as Gish.
A recent study suggested that CL-II phosphorylation was mediated by Gprk2 without providing direct evidence [38]. However, we found that CL-II phosphorylation was not affected by Gprk2 knockdown or overexpression (S3 Fig). In addition, a recombinant GRK failed to phosphorylate a Smo fragment containing the CL-II site in an in vitro kinase assay [24]. Instead, our previous study indicated that Gprk2 phosphorylated Smo at Ser/741/Thr742 (GPS1) and Ser1013/Ser1015 (GPS2) and that mutating these sites to nonphosphorable Ala in the SmoSD123 background attenuated the activity of this constitutively active form of Smo [24]. Here, we further demonstrated that Smo with Gprk2 site mutated (SmoGPSA) failed to activate en in smo mutant clones located near the A/P boundary (S6 Fig), suggesting that Gprk2-mediated Smo phosphorylation is also required for high-levels of Hh signaling activity. Interestingly, both Gish-and Gprk2-mediated phosphorylation was regulated by PKA (Fig 4) [24], which phosphorylated Smo at S667, S687, and S740 [19][20][21]. Indeed, mutating the three PKA sites to Ala (SmoSA123) diminished while converting the PKA sites and adjacent CK1 sites to phospho-mimetic residues (SmoSD123) promoted CL-II phosphorylation. Although SmoSD123 could activate both lowand high-threshold Hh target genes when expressed at high levels, expression of SmoSD123 at close to physiological levels resulted in low to intermediate levels of Hh pathway activity ( Fig  6A-6A") [19]. Phospho-mimetic mutations at Gprk2 or both Gprk2 and Gish sites in the SmoSD123 background resulted in a progressive increase in its activity (Fig 6D-6D"). Hence, Hh-induced phosphorylation of Smo at PKA sites may confer low to medium levels of pathway activation, while high levels of pathway activity require further phosphorylation by Gish and Gprk2, which is "primed" by PKA-mediated phosphorylation. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation requires higher levels of Hh or longer exposure to the same levels of Hh than PKA site phosphorylation (Fig 4I and 4J), suggesting graded Hh signals may differentially regulate PKA-and Gish-mediated phosphorylation of Smo to progressively increase Smo activity (Fig 9).
How does PKA regulate CL-II phosphorylation? Our previous study revealed that Smo adopts a closed conformation in which its C-terminal region folds back and lies in close proximity to its third intracellular loop, whereas Hh-induced phosphorylation at PKA/CK1 sites promotes an open conformation [12]. It is possible that the Gish-binding pocket is masked when Smo C-tails adopt the closed conformation, whereas phosphorylation-mediated conformation switch exposes the Gish-binding pocket. Indeed, we found SmoSD123 exhibited high basal binding to Gish, whereas SmoSA123 lost Hh-stimulated binding to Gish (Fig 7G). However, Hh could further stimulate the binding of Gish to SmoSD123 and increase CL-II phosphorylation in SmoSD123 (Figs 4H and 7G), implying that Hh could regulate Smo/Gish association and CL-II phosphorylation through additional mechanism(s). We noticed that Hh increased the binding of Gish to SmoΔ650 (Fig 7F), which lacks the C-terminal region, including the three PKA sites, suggesting that Hh signaling may induce a conformation change in the transmembrane helixes of Smo, similar to those observed for GPCRs in response to agonist stimulation [47], to expose the juxtamembrane binding site for Gish.
Hh-induced phosphorylation of Smo by PKA promotes its cell surface accumulation [19], which may also contribute the elevated CL-II phosphorylation upon Hh stimulation. In this regard, it is interesting to note that Gish is associated with the plasma membrane through its C-terminal palmitoylation. Cell surface accumulation of Smo will render its close proximity with the plasma membrane-associated Gish and facilitate its binding to Gish. Interestingly, we found that vertebrate CK1γ is localized to the primary cilium depending on its C-terminal palmitoylation. Deleting the palmitoylation site (CK1γ-ΔC) prevented its ciliary localization, its association with mammalian Smo (mSmo), and its ability to phosphorylate mSmo and promote Shh pathway activity. Furthermore, depleting primary cilia by expressing a dominant negative for of Kif3b (DN-Kif3b) also affected CK1γ Smo interaction and phosphorylation by CK1γ. Taken together, these results suggest that only ciliary localized but not the cytosolic CK1γ is capable of phosphorylating Smo to promote Shh pathway activation. As Shh induces mSmo ciliary accumulation, we propose that ciliary accumulation of mSmo may facilitate its binding to and phosphorylation by CK1γ, which contributes to optimal Smo activation (Fig 9). Because abnormal Smo activation contributes to many types of human cancer, the finding that CK1γ is a conserved positive regulator of Hh signaling raises an interesting possibility that interfering with the interaction between CK1γ and Smo may serve as a strategy for cancer treatment.