PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail

In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo.


Author Summary
The Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis. A critical step in Hh signal transduction is the inhibi-

Introduction
The Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis [1][2][3]. In Drosophila, the Hh signal is transduced through a receptor system at the plasma membrane, which includes the receptor complex Patched-Interference Hh (Ptc-Ihog) and the signal transducer Smo [4][5][6]. Binding of Hh to Ptc-Ihog relieves the Ptc-mediated inhibition of Smo, which allows Smo to activate the cubitus interruptus (Ci)/Gli family of zinc finger transcription factors and thereby induce the expression of Hh target genes such as decapentaplegic (dpp), ptc, and engrailed (en) [7,8]. Over the last 30 years, many Hh pathway components have been identified, including those that control transmission, propagation, receipt, and transduction of the Hh signal. However, it is still unclear how Ptc inhibits Smo to block the activation of the Hh pathway and how Ptc inhibition of Smo is relieved by Hh stimulation. It is unlikely that Ptc inhibits Smo by direct association [9,10], as the inhibition occurs even when Smo is present in 50-fold molar excess of Ptc, and substochiometric levels of Ptc can repress Smo activation [10,11]. These findings suggest that the inhibition process is catalytic [10]. The involvement of small molecules, rather than a protein ligand, has been proposed: Ptc may inhibit the production of positive regulators or promote the synthesis of inhibitory molecules [10].
Smo, an atypical G protein-coupled receptor (GPCR), is essential in both insects and mammals for transduction of the Hh signal [8,12,13]. The activation of Smo appears to be one of the most important events in Hh signaling. Hh induces cell surface accumulation and phosphorylation of Smo [9] by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1) [14][15][16], casein kinase 2 (CK2) [17], G protein-coupled receptor kinase 2 (Gprk2) [18], and atypical PKC (aPKC) [19]. These phosphorylation events activate Smo by inducing a conformational change [20] to promote Smo interaction with the Costal2-Fused (Cos2-Fu) protein complex [21][22][23]. It is believed that Hh-induced phosphorylation counteracts the autoinhibition imposed by arginine clusters in the Smo C-terminal tail (C-tail), which induces an open conformation that promotes the dimerization of Smo proteins [1,20]. Similar to other GPCRs, Smo cell surface accumulation is controlled by endocytic trafficking mediated by ubiquitination [24,25]. In mammals, Hh signal transduction depends on the primary cilium, and Smo accumulation in the cilium is required for Smo activation [26][27][28]. Therefore, the cilium represents a signaling center for the Hh pathway in mammals [29]. Phosphorylation by multiple kinases promotes the ciliary localization of mammalian Smo [30], but it remains unclear how Smo cell surface or ciliary accumulation and intracellular trafficking are controlled. A previous study has shown that mutation in INPP5E, a lipid 5-phosphotase, results in signaling defects in primary cilium [31], indicating a role for phospholipids to regulate the function of cilium. In the preparation of this manuscript, two studies found that phospholipids regulate ciliary protein trafficking [32,33]; however, it is unknown whether and how phospholipids directly regulate Smo.
A very well characterized system for studying Hh signaling is the Drosophila wing disc. Hh proteins expressed and secreted from the posterior (P) compartment cells act on neighboring anterior (A) compartment cells located adjacent to the A/P boundary to induce the expression of Dpp [34,35]. As a morphogen, Dpp diffuses bidirectionally into both the A and P compartments to control the growth and patterning of cells in the entire wing [36][37][38]. Other genes, including en and ptc, are also induced by Hh to specify cell patterning at the A/P boundary [39,40]. Expression of dpp monitors the low levels of Hh activity, and ptc expression indicates higher levels of Hh activity, whereas en induction appears to be an indicator of the highest doses of Hh signaling activity [39]. The transcription factor Ci is only expressed in A compartment cells that receive the Hh signal.
In this study, we found that Hh stimulation increases the levels of phosphatidylinositol 4-phosphate (PI(4)P) in both wing discs and cultured cells. We further found that PI(4)P activates Smo by promoting Smo phosphorylation. Mechanistically, we identified an arginine motif in the Smo C-tail that is responsible for the interaction of Smo with PI(4)P. Arginine to alanine mutation abolishes, whereas arginine to glutamic acid mutation elevates, Smo activity. We also found that, in addition to the kinase activity of Gprk2, its pleckstrin homology (PH) domain increases PI(4)P in wing discs and is required for Gprk2 to fully function in Hh signaling. The findings that Ptc interacts with PI(4)P and that Ptc inactivation increases the levels of PI(4)P indicate that PI(4)P acts downstream of Ptc to activate Smo in the Hh signaling cascade. Finally, we show that PI(4)P promotes phosphorylation and ciliary accumulation of mouse Smo (mSmo) in mammalian cells, and that PI(4)P prevents the ciliary accumulation of mouse Ptc1 and Ptc2. Taken together, our findings suggest that PI(4)P acts as a special small molecule shuttling between Ptc and Smo to modulate Hh responses.

Hh Promotes the Production of PI(4)P
In an effort to identify novel regulators in Hh signaling, we collected RNAi lines from the Vienna Drosophila Resource Center (VDRC) when the library was available and screened for kinases, phosphatases, and E3 ubiquitin-protein ligases using the wing-specific MS1096-Gal4. We tested selected RNAi lines for the ability to modify the phenotype of Smo PKA12 , a weak dominant negative form of Smo that results in a reproducible wing phenotype with partial fusion between Vein 3 and Vein 4 when combined with the C765-Gal4 (S1D Fig), which represents a very sensitive genetic background for screening Smo regulators [19,25,41]. One of the "hits" was Stt4 kinase, the yeast homolog of PI4KIIIalpha required for the generation of PI(4)P. We found that, although knockdown of Stt4 alone did not produce any change in the wild-type wing (S1B Fig), Stt4 RNAi combined with Smo PKA12 expression enhanced the fusion of Vein 3 and Vein 4 (S1E Fig). In contrast, inactivation of Sac1 phosphatase, which dephosphorylates PI (4)P to phosphatidylinositol (PI), attenuated the fusion phenotype (S1F Fig). These results suggest that Stt4 and Sac1 regulate the activity of Smo in the wing. Consistently, Sac1 RNAi partially rescued the abdominal cuticle loss caused by Hh RNAi, although Sac1 RNAi alone did not show any cuticle phenotype (S1G-S1J Fig). A recent study established a genetic link between Smo and Stt4-Sac1 [42]; however, the molecular mechanisms are unclear.
Smo accumulates in P compartment cells as well as A compartment cells near the A/P boundary ( Fig 1A) [9,15]. We found that the level of PI(4)P is elevated in the A compartment cells that abut the A/P border (Fig 1B), suggesting that Hh induces the accumulation of both Smo and PI(4)P in these cells. Consistently, the accumulation of Smo in Drosophila embryo indicates the A/P boundary defined by Ci staining. Green box indicates the region for density analysis that is shown on the right, with a yellow line indicating the A/P boundary. (B) A WT disc was stained for Ci and PI(4)P. Arrow indicates PI(4)P accumulation in A compartment cells near the A/P boundary, and arrowhead indicates PI(4)P accumulation in P compartment cells. Dashed yellow lines indicate the A/P boundary defined by Ci staining. Green box indicates the region for density analysis using ImageJ software (NIH, version 1.48v) that is shown on the right with a yellow line indicating the A/P boundary. All wing imaginal discs shown in this study were dissected from third instar larvae with different genotypes and are shown with anterior on the left and ventral on the top. (C) Effect of Hh treatment on PIP content. S2 or NIH3T3 cells were stimulated with either 60% Hh-conditioned medium or Shh protein (5nM) before lipid extraction. A Thermo TSQ Vantage triple-quad mass spectrometer coupled with a Shimadzu HPLC as the front-end separation was used for the MRM measurement. * p < 0.01 versus time 0 (Student's t test). (D) Left panel: S2 cells were treated with Stt4 dsRNA or Sac1 dsRNA, followed by treatment with control medium, 30% Hh-conditioned medium (Hh + ), or 60% Hhconditioned medium (Hh ++ ). PI(4)P was detected by ELISA. * p < 0.01 versus control (first column); † p < 0.01 versus high level of Hh treatment (third column). Right panel: S2 cells were transfected with UAST-Ptc construct or treated with Ptc dsRNA and PI(4)P detected by ELISA. p < 0.01 versus control (first column). (E) S2 cells were treated with the indicated dsRNA followed by treatment with different amounts of Hh-conditioned medium and assayed for ptc-luc reporter activity. * p < 0.01 versus control (first column). † p < 0.01 versus high level of Hh (third column). † † p < 0.05 versus high level of Hh (third column). The efficiency of RNAi is shown in S3C and S3D Fig. (F) S2 cells were transfected with indicated constructs, treated with 60% Hh-conditioned medium, and assayed for ptc-luc activity. * p < 0.05 versus control. † p < 0.001 versus Hh alone. In these experiments, S2 cells stably expressing tub-Ci were used as they have full responsiveness to Hh stimulation. The expression of HA-Inp54p, Inp54p D281A , and IpgD are shown in the right panel by direct western blot with the anti-HA antibody, using lysates from cells expressing the indicated HA-tagged constructs. GFP was used as a transfection and loading control. The underlying data of panels C-F can be found in S1 Data. correlated with the accumulation of PI(4)P (S2A- S2C Fig). We further found that PI(4)P levels were increased by the expression of Ci -3P and Smo SD123 , the constitutively active forms of Ci  and Smo, respectively (S2D and S2E Fig). To accurately measure and quantify the absolute concentration of PIP in cells, we established a mass spectrometry-based multiple reaction monitor (MRM) assay and examined whether Hh indeed induces the production of PIP. Based on a detailed method published recently for quantifying PIP 2 (PI(3,4)P 2 , PI(3,5)P 2 , PI(4,5)P 2 ), and PIP 3 (PI(3,4,5)P 3 ) [43], we optimized the conditions to examine PIP lipids. Trimethylsilyl diazomethane was used to protect the phosphate groups, which allowed for more efficient ionization of the methylated PIP species and a marked improvement in the sensitivity of the assay. We found that treatment of S2 cells with 60% Hh-conditioned medium [44] induced the formation of PIP in a timely manner (Fig 1C, left panel). Consistent with this, treatment of NIH3T3 mouse fibroblasts with mouse sonic Hh N-terminus (ShhNp) [30] stimulated the production of PIP (Fig 1C, right panel). Total PIP was quantified, since this assay was unable to distinguish PI(4)P from PI(3)P and PI(5)P.
To further characterize the regulation of PI(4)P by Hh, we used an enzyme-linked immunosorbent assay (ELISA) and found that Hh stimulated the production of PI(4)P in S2 cells in a concentration-dependent manner ( Fig 1D, left panel). In addition, knockdown of Stt4 downregulated, whereas knockdown of Sac1 upregulated, the production of PI(4)P ( Fig 1D, left panel), suggesting that the Hh-regulated formation of PI(4)P was mediated by Stt4 and Sac1. We further found that overexpression of Ptc prevented the production of PI(4)P, whereas RNAimediated knockdown of Ptc elevated production ( Fig 1D, right panel), suggesting that Ptc regulates the levels of PI(4)P. To delineate the involvement of PI(4)P in Hh signaling, we used a ptc-luciferase (ptc-luc) reporter assay to monitor the activity of Hh signaling [44] and found that Hh-induced ptc-luc activity was suppressed by RNAi of Stt4 but elevated by RNAi of Sac1 ( Fig 1E). Furthermore, treatment with PI(4)P and the expression of Inp54p (a PI(4,5)P 2 -specific phosphatase to produce PI(4)P) enhanced, whereas IpgD (converts PI(4,5)P 2 into PI(5)P) suppressed, the basal and Hh-induced ptc-luc activity (Figs 1F and S3A). As a control, Inp54p D281A phosphatase-dead mutant had no effect on ptc-luc activity ( Fig 1F). These data suggest that PI(4)P is a specific phospholipid that regulates Hh signaling in cultured S2 cells.

PI(4)P Stimulates the Phosphorylation and Accumulation of Smo
The PH domain is a known phosphoinositide-binding module that is important for signal transduction by sensing alterations in the membrane lipid composition. To visualize PI(4)P pools in wing discs, we used an RFP-PH OSBP reporter that contains two copies of the PH domain from the oxysterol binding protein (OSBP), which is known to specifically bind PI(4)P [45]. In wing discs, expression of RFP-PH OSBP accumulated PI(4)P ( Fig 2B) and Smo (Fig 2C), compared to the expression of RFP alone (Fig 2A). In cultured S2 cells, treatment with PI(4)P enhanced Smo activity, indicated by an elevated ptc-luc reporter activity (S3B Fig), thus prompting the question of whether PI(4)P regulates Smo phosphorylation, since phosphorylation promotes Smo activation. Indeed, we found that PI(4)P, but not other phospholipid forms, increased the levels of basal and Hh-induced Smo phosphorylation detected by a phospho-specific antibody (SmoP) [44] recognizing phosphorylation within the second PKA/CK1 cluster ( Fig 2D). In addition, PI(4)P treatment induced Smo phosphorylation to a lesser extent compared to Hh treatment, and the combination of Hh and PI(4)P induced hyperphosphorylation of Smo ( Fig 2E). Consistently, treatment with PI(4)P induced mSmo phosphorylation in cultured NIH3T3 cells, which was detected by a phospho-specific antibody (PS1) [30] recognizing mSmo phosphorylation at the first CK1/GRK cluster (Fig 2F). In an in vitro kinase assay using glutathione S-transferase (GST)-Smo fusion protein containing Smo amino acids 656-755, we found that Smo phosphorylation by PKA and CK1 kinases was enhanced by the addition of PI(4)P, but not PI(4,5)P 2 or PIP 3 (Fig 2G), suggesting that PI(4)P directly regulates the phosphorylation of Smo. In support of this notion, we found that Smo interacted with the PH domain from OSBP, and that this interaction was enhanced by the treatment with either PI(4) P or Hh (Fig 2H and 2I).

PI(4)P Regulates Smo Phosphorylation through Direct Interaction with an Arginine Motif
It is possible that PI(4)P directly interacts with Smo and facilitates Smo interaction with the PH domain of OSBP. To test this, we used a solid-phase lipid-binding assay and found that purified full-length Myc-Smo WT strongly associated with PI(4)P, and weaker binding to PI(5)P was detected as well ( Fig 3A, left column). Because the level of PI(5)P is much lower than that of PI(4)P in cells [46], PI(4)P is likely the primary lipid that binds Smo. We further found that   [20]. Surprisingly, using GST-Smo 656-755 in the solid-phase lipid-binding assay, we found that Smo WT strongly interacted with PI(4)P; however, an Arg to Ala mutation (GST-Smo RA4 ) abolished this interaction (Fig 3H), suggesting PI(4)P interacts with only the fourth arginine cluster. In support of this finding, mutation in the fourth arginine cluster (R4) was sufficient to block PI(4)P binding in a PI(4)P beads pull-down assay ( Fig 3I). To further characterize R4, we generated the Arg to Ala mutation in  Fig 3Q). These data suggest that Smo activity is compromised by R>A mutation in the fourth arginine motif. In contrast, we found that R>E mutation (Smo RE4 ), which mimics negative charges caused by PI(4)P binding, elevated Smo activity to induce higher levels of dpp-lacZ expression in the wing disc ( Fig 3P) and higher levels of ptc-luc activity, but had no responsiveness to PI(4)P stimulation (S4E Fig). Taken together, our findings suggest that the fourth arginine motif is required for Smo activation.
were then incubated with PI(4)P beads, followed by western blot with the anti-Myc antibody to examine the bound Myc-tagged proteins. (K) S2 cells were transfected with the indicated constructs and treated with Hhconditioned medium and/or PI (4) In comparison, fusion of CFP and YPF to the third intracellular loop and C-tail, respectively, did not change the activity of Smo [20]. Our findings suggest that PI(4)P binds Smo in a positiondependent manner.

The PH Domain of Gprk2 Mediates Smo Regulation by PI(4)P
Considering that the PH domain of OSBP interacts with and activates Smo, we wondered whether a PI(4)P transport protein (PITP) facilitates the interaction between PI(4)P and Smo, since Smo itself does not contain a PH domain. We used RNAi lines from the VDRC to screen a total of 15 typical PH domain-containing PITPs in the fly genome for their ability to modulate Hh phenotypes; inactivation of these proteins by RNAi did not affect Smo accumulation in wing discs, although RNAi of some candidate PITPs modified the wing phenotype of C765-Smo PKA12 (S1 Table). Interestingly, all Gprks contain a PH domain in their C-terminus, and this domain contributes to agonist-dependent translocation by facilitating interaction with lipids and other membrane proteins [47,48]. We next investigated whether the C-terminus PH domain of Gprk2 was important for its role in Hh signal transduction. Wild-type Gprk2 fully rescued en expression in gprk2 mutant cells ( Fig 4A). However, deletion of the PH domain in the Gprk2 C-tail (Grpk2 ΔC ) abolished its ability to rescue en expression (Fig 4B), whereas replacing the PH domain in Gprk2 with the PH domain from OSBP (Gprk2-PH OSBP ) restored this ability (Fig 4C). This is consistent with our previous finding that Gprk2KM, a kinase-dead form of Gprk2, has a kinase activity-independent role in regulating Smo [18]. These findings suggest that the PH domain is required for Gprk2 to fully function in transducing the Hh signal. In support of these results, Gprk2, Gprk2 ΔC , and Gprk2-PH OSBP , but not Gprk2KM, were able to phosphorylate mSmo in vitro (Fig 5A), indicating that the removal or replacement of the PH domain does not affect the kinase activity. Thus, the function of the Gprk2 PH domain likely accounts for the kinase-independent role of Gprk2 in Smo regulation.
Because both Gprk2 transcription and Gprk2 protein expression are upregulated by Hh signaling, and Gprk2 is enriched at the A/P boundary [18,49], we hypothesize that, in addition to promoting the production of PI(4)P, Hh may regulate PI(4)P accumulation by enhancing the expression of Gprk2 as the endogenous carrier for PI(4)P. To examine the ability of Gprk2 to enrich PI(4)P in vivo, we knocked down Gprk2 in the wing disc and found that the levels of PI (4)P were decreased (S5A Fig). We also overexpressed Gprk2 or Gprk2 ΔC and found that the expression of Gprk2 elevated the levels of both Smo and PI(4)P (Fig 4D and 4E), whereas the expression of Gprk2 ΔC had no effect (Fig 4G and 4H). Similar to RFP-PH OSBP (Fig 2B and 2C), overexpression of PH Gprk2 resulted in increased PI(4)P and Smo accumulation (S5B and S5C  Fig). In addition, Gprk2 and PI(4)P were largely localized at the cell surface ( Fig 4F and 4F 0 ), whereas Gprk2 ΔC was cytosolic (Fig 4I). These results suggest that the PH domain of Gprk2 is required for the enrichment of PI(4)P in vivo by localizing Gprk2 at the cell surface.
To further characterize the kinase activity-independent role of Gprk2 in regulating Smo, we examined Gprk2-regulated Smo phosphorylation in cultured S2 cells. We found that RNAi targeting the coding region of Gprk2, but not OSBP, attenuated PI(4)P-induced Smo phosphorylation detected by the anti-SmoP antibody (Fig 5C). RNAi targeting the 3 0 -UTR region of Gprk2 consistently inhibited Smo phosphorylation (Fig 5D, lane 4, top panel). We found that the expression of HA-Gprk2 or HA-Gprk2-PH OSBP rescued Smo phosphorylation inhibited by RNAi of Gprk2 3 0 -UTR but the expression of HA-Gprk2 ΔC did not (Fig 5D), suggesting that the PH domain is responsible for Gprk2 to promote Smo phosphorylation increased by PI(4)P. We also found that deletion of the PH domain decreased the Gprk2-PI(4)P interaction in the PI(4)P beads pull-down assay ( Fig 5E). Moreover, the PH domain of Gprk2 (PH Gprk2 ) interacted directly with PI(4)P; mutation of arginine (PH Gprk2RA ) or phenylalanine (PH Gprk2FA ) abolished this interaction (Fig 5F). Finally, similar to the PH OSBP interaction with Smo ( Fig 2H  and 2I), the PH Gprk2 interaction with Myc-Smo WT was increased by Hh and PI(4)P treatments in cultured S2 cells (Fig 5G and 5H). Taken together with the observation that deletion of the PH domain does not alter the kinase activity of Gprk2 in vitro ( Fig 5A) and in cultured NIH3T3 cells (Fig 5B), our findings suggest that the Gprk2 PH domain plays a positive role in mediating Smo regulation by PI(4)P.

Hh Treatment Increases Smo-PI(4)P Interaction and Decreases Ptc-PI (4)P Interaction
The finding that expression of the PH domain from OSBP accumulated PI(4)P (Fig 2B) prompted the notion that an endogenous protein may attract PI(4)P away from Smo in the absence of Hh. Ptc contains a sterol-sensing domain (SSD) and has structural similarity to the resistance, nodulation, division (RND) family of bacterial proton gradient-driven  [50]. SSD was first identified in proteins implicated in cholesterol metabolism but is now more broadly associated with vesicle trafficking. The Ptc SSD is essential for suppression of Smo activity [51], and mutations of SSD abrogate the Ptcmediated repression of Smo, although these mutations do not compromise either binding or internalization of Hh [10,52]. It is possibe that the Ptc SSD controls the influx or the efflux of PI(4)P or attracts PI(4)P away from Smo. To test this hypothesis, we generated three vectors: HA-tagged wild-type full-length Ptc (HA-Ptc WT ), HA-tagged Ptc lacking its SSD domain (HA-Ptc ΔSSD ), and HA-tagged SSD domain (HA-SSD). We transfected S2 cells with these constructs and evaluated the ability of each to interact with PI(4)P. When expressed in S2 cells, all proteins were expressed at low levels, detected only after immunoprecipitation (Fig 6A, top  panel). We found that HA-Ptc WT and HA-SSD strongly bound PI(4)P, whereas HA-Ptc ΔSSD did not bind (Fig 6A, lower panel).
To further determine whether the SSD domain from Ptc directly interacts with PI(4)P, we used the solid phase lipid-binding assay similar to that used for detecting Smo binding. We found that the SSD fragment protein purified from bacteria strongly associated with PI(4)P, but not with PIP 2 or PIP 3 phospholipids (Fig 6B), suggesting that the interaction between SSD and PI(4)P in the lipid beads protein pull-down assay is direct. The SSD association with PI(3) P or PI(5)P (Fig 6B) suggests that the expression of a single SSD domain may lose specificity for interaction, or, alternatively, that such interaction may also promote Ptc regulation of PI(3) P and PI(5)P. We also found a very strong interaction between SSD and phosphatidic acid (PA) or phosphatidylserine (PS) (Fig 6B); these may be nonspecific, as PA and PS binding to short protein fragments has often been considered questionable [53]. Our findings in cultured cells led us to examine the correlation of Ptc and PI(4)P in the wing. We found that mutation of ptc or knockdown of Ptc by RNAi increased PI(4)P levels in the wing disc (Fig 6C and 6D), similar to the observation that Ptc inactivation elevates PI(4)P in the salivary gland [42]. These indicate that the activation of Hh signaling by the inactivation of Ptc elevated the production of PI(4)P. Moreover, we found that the overexpression of Ptc WT also increased the level of PI(4)P (Fig 6E), which is likely due to the ability of Ptc to accumulate PI(4)P. In support of these findings, Ptc ΔSSD overexpression had no effect on regulating the accumulation of Smo, Ci, and PI (4)P in wing discs.
In addition to promoting the production of PI(4)P, Hh may also regulate the pools of PI(4) P between Smo and Ptc. To test this hypothesis, we purified Smo and Ptc proteins from S2 cells treated with Hh-conditioned medium or control medium and accessed the protein interaction with PI(4)P. As shown in Fig 6E, the level of PI(4)P-bound Smo was increased by the treatment of Hh (Fig 6F, left panel). In contrast, the level of PI(4)P-bound Ptc was decreased by Hh treatment (Fig 6F, right panel). These data indicate that Hh treatment releases PI(4)P from Ptc, suggesting an additional layer of regulation beyond Hh promoting PI(4)P production.
It would be interesting to understand how Hh regulates PI(4)P. However, we found that Hh treatment did not significantly change the mRNA levels of Stt4 and Sac1 (S6A Fig). In addition, Hh did not change the protein levels of the overexpressed Stt4 and Sac1 in P compartment cells of the wing disc (S6B and S6C Fig). Hh also did not regulate the accessibility of the Stt4/Sac1 to Smo or Ptc in an immunoprecipitation assay with S2 cells. To examine whether Hh regulates the activity of Stt4 or Sac1, or both, we carried out in vitro kinase/phosphatase assays using purified Stt4 and Sac1 combined with PI substrate. We found that the phosphorylation of PI was enhanced when using Stt4 from cells treated with Hh ( Fig 6G, lane 3, compared to lane 2, left panel). In addition, Sac1 from cells treated with Hh had less activity to dephosphorylate the constitutive PI phosphorylation (Fig 6G, lane 3, compared to lane 2, right panel). These data suggest that Hh elevates the activity of Stt4 and inhibits the activity of Sac1. Ptc SSD domain interacts with PI(4)P. (A) S2 cells were transfected with the indicated HA-tagged Ptc constructs and then split in half. One half was used to detect Ptc protein by immunoprecipitation with the anti-HA antibody and western blot analysis using the anti-HA antibody. The second half was used to detect PI(4)P bound Ptc by lipid beads protein pull-down assay using PI(4)P beads and western blot using anti-HA antibody. The SSD domain is responsible for Ptc binding to PI(4)P. (B) Ptc binding in a protein:lipid overlay assay. Lipids dotted strips were incubated with bacterially expressed His-tagged SSD (His-SSD), and bound-SSD was detected with an anti-His antibody. (C) A wing disc bearing ptc-/-mutant clones was immunostained for PI(4)P and GFP. Arrows indicate the mutant clone marked by the lack of GFP expression. (D-E) Wing discs expressing either PtcRNAi #28795 or HA-Ptc WT were stained for PI(4)P and Ci. Arrows indicate the accumulation of PI(4)P by RNAi and overexpression of Ptc. (F) S2 cells were transfected with either Myc-Smo WT or Myc-Ptc WT , treated with Hh-conditioned medium or control medium, and immunoprecipitated with the anti-Myc antibody. Purified proteins were then incubated with PI(4)P beads followed by western blot with the anti-Myc antibody to examine the bound Myc-tagged proteins. Hh treatment increases the level of PI(4)P-bound Smo and decreases the level of PI(4)P-bound Ptc. Similarly, Hh treatment promotes Smo-PI(4)P interaction and inhibits Ptc-PI(4)P interaction in an independent assay using PI(4)P liposomes. (G) In vitro kinase assay (left) and in vitro phosphatase assay (right) were carried out to examine Stt4 and Sac1 enzymatic activity regulated PI(4)P Promotes mSmo Activation and Localization in the Cilium Next, we wondered whether PI(4)P plays a role in regulating mSmo, because PI(4)P induces mSmo phosphorylation (Fig 2F). We first tested different phospholipids for their effects in activating mSmo and found that, similar to Shh, PI(4)P treatment elevated mSmo activity as monitored by a Gli-luc reporter (Fig 7B). In contrast, PIP 2 and PIP 3 treatment had no effect on mSmo activity. In addition, the activity of the constitutively active form of mSmo (mSmo SD ), which mimics mSmo phosphorylation by GRK2 and CK1 [30], was further increased by PI(4) P ( Fig 7B). Consistently, Drosophila Smo SD123 activity was increased by PI(4)P (S4E Fig). Similar to Drosophila Smo, mSmo contains arginine clusters in its C-tail (Fig 7A) [20]. We next examined whether the arginine motif(s) were responsible for regulation of mSmo by PI(4) P. As shown in Fig 7C, PI(4)P treatment increased Gli-luc reporter activity, although to a lesser extent compared to Shh treatment in the control group of NIH3T3 cells. PI(4)P and Shh treatment consistently increased Gli-luc activity when cells were transfected with mSmo WT ( Fig  7C). However, R>A mutations in R3 and R4 arginine clusters (mSmo RA3 and mSmo RA4 , respectively) attenuated the increased activity noted with PI(4)P, and mutations in both R3 and R4 (mSmo RA34 ) completely blocked the effect of PI(4)P on mSmo activation (Fig 7C). These data suggest that R3 and R4 are responsible for the regulation of mSmo by PI(4)P.
Phosphorylation promotes ciliary accumulation of mSmo, which correlates with pathway activation [30], but the molecular mechanisms that control Smo ciliary accumulation are poorly understood. mSmo WT was found in about 5% of cilia, and Shh treatment increased Smo WT accumulation in 75% of cilia (Fig 7D and 7E) [30]. Treatment with PI(4)P induced mSmo WT accumulation in 47% of cilia (Fig 7D and 7E), which was correlated with changes in mSmo WT phosphorylation ( Fig 2F) and activity (Fig 7B) induced by PI(4)P. In contrast, mSmo RA34 had no response to PI(4)P treatment (4% of ciliary accumulation by PI(4)P treatment) (Fig 7D and 7E), although it had a low response to Shh stimulation (from 3% to 21% of ciliary localization). Consistent with these findings, mSmo RA34 had much lower activity and much less responsiveness to Shh stimulation in a previous study [20]. Our findings suggest that R3 and R4 clusters are responsible for PI(4)P-associated binding and activation of mSmo.
To determine whether Hh regulates the production of PI(4)P in vertebrate systems, using the ELISA assay combined with the anti-PI(4)P antibody, we examined the levels of PI(4)P in ptc1 mutant mouse embryonic fibroblasts (MEFs) and found that, compared to control MEFs, ptc1 MEFs had significantly increased PI(4)P (Fig 7F). Consistently, Shh treatment increased, whereas Smo inhibitor decreased, the levels of PI(4)P (S7A Fig), indicating that Hh signaling activity promotes PI(4)P production in cultured cells. We further investigated the ciliary localization of Ptc1 and Ptc2 and found that the ciliary localization of both Ptc1 and Ptc2 was decreased by Shh treatment or PI(4)P treatment (Fig 7G), which was consistent with the previous study that Hh inhibits the ciliary localization of Ptc1 [54]. Similar to Drosophila Ptc interaction with PI(4)P, we found that both Ptc1 and Ptc2 interacted with PI(4)P in the lipid beads protein pull-down assay (Fig 7H). These observations indicate a consistent regulation of Smo and Ptc by PI(4)P in Drosophila and mammalian systems. To incorporate the findings in this study and the findings published recently [32,33,42], we proposed a model in which Smo phosphorylation and ciliary accumulation is regulated by PI(4)P (Fig 8).
by Hh. Stt4 and Sac1 were purified from S2 cells transfected with Flag-Stt4 or HA-Sac1 with the treatment of either Hh-conditioned medium or control medium. The substrates were chromatographed on oxalate-pretreated silica gel plate and visualized by autoradiography (see S1 Methods for details).  Gli-luc reporter assay in NIH3T3 cells transfected with blank vector (control), mSmo WT , or mSmo SD and treated with SAG, Shh, or individual lipid. Carrier 3 served as the treatment control. * p < 0.01 versus carrier. † p < 0.001 versus carrier. † † p < 0.01 versus carrier in the mSmo SD group. (C) Gli-luc assay in NIH3T3 cells transfected with blank vector (control) or each mSmo construct and treated with either PI(4)P or Shh. * p < 0.01 versus control plus PI(4)P. ** p < 0.001 versus control plus PI(4)P. † p < 0.01 versus control plus Shh. † † p < 0.001 versus control plus Shh. Note, the overexpressed mSmo induced higher levels of Hh pathway activation, compared to Shh treatment, which might be due to the Shh peptides used or the high level expression of the transfected mSmo. (D) NIH3T3 transfected with EGFP-tagged mSmo WT or mSmo RA34 and treated with Shh or PI(4)P were immunostained to show the expression of Acetylated (Ac)-tubulin (red; primary cilium), GFP (green; mSmo), and PS1 (blue; phosphorylated mSmo). Images in the inserts are enlarged views with shifted overlays to show the ciliary localization of mSmo. About 100 ciliated cells were counted for each set. (E) Quantification of ciliary localization of infected mSmo WT and mSmo RA34 as indicated by the percentage of GFP + cilia shown in (D). (F) ELISA assay was carried out to examine the levels of PI(4)P in either control MEFs or ptc1-/-MEFs. Bars

Discussion
Hh signal transduction has been widely studied; however, the longstanding questions of how Ptc inhibits Smo activity and how Hh promotes Smo phosphorylation and activation remain. These issues constitute a primary focus of this study. A genetic analysis indicates that inactivation of Stt4 downregulates Smo accumulation, whereas knockdown of Sac1 by RNAi elevates Smo levels [42], suggesting the involvement of phospholipids in Hh signal transduction. Nevertheless, the mechanisms by which phospholipids regulate Hh signaling remain unknown. In this study, we identified and characterized a direct role for PI (4)

Kinase Activity-Independent Role of Gprk2 in Hh Signaling
To explore the possible involvement of a PITP protein to facilitate the interaction between Smo and PI(4)P, we unexpectedly found that the PH domain of Gprk2 is responsible for the accumulation of PI(4)P that activates Smo. It has been shown that Gprk2 is positively involved in Hh signaling by directly phosphorylating Smo C-tail [18]. In addition, Gprk2 forms a dimer/ oligomer and binds Smo C-tail in a kinase activity-independent manner to promote Smo dimerization and activation [18]. However, how Gprk2 promotes Smo dimerization and activation is unclear. In this study, we found that the function for Grpk2 PH domain to activate indicate three independent repeats. * p < 0.001. (G) NIH3T3 cells transfected with YFP-Ptc1 or GFP-Ptc2 and treated with Shh or PI(4)P were immunostained to examine the ciliary localization of Ptc1 and Ptc2 (see S7B and S7C Fig for example images). About 100 ciliated cells were counted for each set. The relative small numbers of Ptc1 and Ptc2 in the cilia are likely due to transient transfection and the expression levels of the protein; however, statistical analysis data was collected from the same sets of cells. (H) Myc-Ptc1 or Myc-Ptc2 was transfected into NIH3T3 cell, purified by immunoprecipitation with the anti-Myc antibody, and incubated with PI(4)P beads or control beads followed by western blot with the anti-Myc antibody to examine the bound Myc-tagged proteins. The underlying data of panels B-C and E-G can be found in S1 Data.  Models are based on this study and studies from recent publications. In the absence of Hh, Stt4 has low activity, whereas Sac1 has high activity, resulting in low levels of PI(4)P that interact with Ptc. In the vertebrate cilium, Ptc1/2 and Grp161 localize in the cilium to inhibit Hh signaling. PI(4,5)P 2 accumulates in the cilium, but not PI(4)P. Upon Hh stimulation, PI(4)P production is increased and PI(4)P is also released from Ptc, allowing PI (4) Smo is independent of its kinase activity (Fig 5A and 5B) and that the PH domain of Gprk2 is responsible for enriching PI(4)P that promotes Smo phosphorylation and dimerization. There are instances in which the binding of lipids to the PH domain promotes dimerization of the protein [55,56], raising the possibility that PI(4)P interaction with the PH domain of Gprk2 promotes its dimerization. Taken together, our findings suggest that the function of PH domain in Gprk2 accounts, at least in part, for the kinase activity-independent role of Gprk2 in Hh signaling, a deeper mechanism for Smo activation by Gprk2.

The Release of PI(4)P from Ptc in Response to Hh Stimulation
In the absence of Hh, Ptc inhibits Smo activity by promoting Smo endocytosis and turnover in intracellular compartments [9]. Ptc likely inhibits Smo catalytically [10], because substochiometric levels of Ptc are able to repress Smo activation [10,11]. Here, we found that Hh promotes the activity of Stt4 and inhibits the activity of Sac1 (Fig 6G), which, at least in part, explains the catalytic regulation.
A previous study proposed a model in which Ptc represses Smo by regulating lipid trafficking; Ptc recruits lipoproteins to endosomes, changing their lipid composition, in order to regulate Smo degradation [57], but the class of lipids remains unidentified. In the presence of Hh, Smo is phosphorylated and accumulates at the cell surface, resulting in protein activation [1,8]. However, it is unknown how Ptc inhibition on Smo is relieved by Hh stimulation. The ability of the Ptc SSD domain to interact with PI(4)P (Fig 6A and 6B) raises the possibility that Ptc may control the pool of phospholipids regulating the accessibility of Smo to PI(4)P. Our finding that Hh treatment decreased the interaction between Ptc and PI(4)P (Fig 6F) suggests the possibility that binding of Hh to Ptc results in a conformational change in Ptc and releases phospholipids. Thus, this study uncovers an additional layer of regulation by indicating the release of PI(4)P from Ptc, which may account for the optimal regulation of Smo.

The Location of Smo Motif Responsible for Binding PI(4)P Is Critical for Changing Smo Conformation
The structure of the Smo N-terminal, including the extracellular cysteine rich domain (CRD), has been characterized [58,59]. Unlike other GPCRs, no ligand-binding function has been identified. It has been shown that Smo-mediated signal transduction is sensitive to sterols and oxysterol derivatives of cholesterol [60][61][62]. However, unlike vertebrate Smo, Drosophila Smo CRD does not interact with oxysterols [63]. In this study, we found that phospholipids activate both vertebrate and Drosophila Smo through binding to the arginine motif in the Smo C-terminus, although the C-tails have sequence divergence among species. Using the protein:lipid overlay assay, we found that PI(4)P directly binds Smo (Fig 3A) and that mutation in the R4 arginine motif abolishes this direct interaction (Fig 3H-3J). Importantly, R>A mutation abolished the activity of Smo (Figs 3L, 3M and S4G). It is likely that binding of PI(4)P to the arginine motif changes Smo conformation, thus allowing kinases to phosphorylate and activate Smo. In support of this notion, PI(4)P binding to specific arginine residues in specific locations is critical for Smo conformational change, because fusion of the PH domain to either the third intracellular loop or the C-terminus attracts PI(4)P to different locations in Smo, thus blocking Smo activation by PI(4)P (S4 Fig).
In this study, we focused on the regulation of Smo by PI(4)P and found that Hh regulates the accessibility of Smo to PI(4)P, evidenced by the Hh-promoted interaction between Smo and PH domain from either OSBP or Gprk2 (Figs 2I and 5E) and by the Hh-enhanced interaction between Smo and PI(4)P (Fig 6F)

Mechanisms for Hh to Promote PI(4)P Production
Hh signaling activity promoted the production of PI(4)P that was detected by the mass-spec assay (Fig 1C), which was a very sensitive approach. However, in a previous study, the overexpression of full-length wild-type Ci did not elevate the accumulation of PI(4)P in wing discs [42]. It is possible that low levels of Hh signaling activity induced by the expression of wildtype Ci are unable to induce detectable changes in PI(4)P accumulation in wing discs. The disc immunostaining with the anti-PI(4)P antibody might not be as sensitive as the mass-spec method. In support, by expressing Smo SD123 or the constitutively active Ci -3P , in which three PKA sites in the phosphorylation clusters were mutated to block Ci processing [64], we found that PI (4)

Phospholipid Quantification by Mass Spectrometry and ELISA Assays
PtdIns lipid extraction and quantification by mass spectrometry were carried out as previously described [43]. Briefly, 3 × 10 7 S2 cells (or 4 × 10 6 NIH3T3 cells) from a 100 mm dish were harvested and washed once with ice-cold PBS and suspended in 340 μL H 2 O and 1500 μL quench mix with 25 ng of 17:0-20:4 PI(4)P (Avanti Polar Lipids, Inc.) in 10 μL methanol as the internal standard. Lipids were extracted with 1450 μL CHCl 3 and 340 μL 2 M HCl and subsequently derivatized with trimethylsilyl diazomethane (Sigma), as previously described [43]. After washing and drying under a stream of nitrogen at room temperature, samples were dissolved in 200 μL methanol. We applied 10 μl of each sample to LC-MS/MS analysis using a Shimadzu LC-20 HPLC and TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher). A Jupiter 5μ C4 300A (50 × 1.0 mm) column (Phenomenex) was used with the multiple reaction monitoring (MRM) transitions described [43]. PIP concentrations were calculated from MRM peak areas and the internal standard and were subsequently normalized to cell number.
For PI(4)P quantification by ELISA assay, similar PtdIns lipid extraction procedures were used without adding the PI(4)P internal control. After washing and drying with nitrogen stream, lipid extracts were dissolved in ethanol and loaded into a microplate, dried under a vacuum, and incubated with 2% BSA in PBS at room temperature for 30 min. Mouse anti-PI(4)P or anti-PI(4,5)P 2 monoclonal antibodies (Echelon Biosciences) were added for 1 h, followed by goat anti-Mouse IgG-HRP (Jackson ImmunoResearch) for 30 min, with 3 PBS washes after each inculation. Finally, chemiluminescence substrate (SuperSignal West Pico, Pierce) was added to the microplate, and luminescence intensity was determined by a luminometer. For the PI(4)P beads pull-down experiments, HA-tagged Gprk2 proteins were expressed in S2 cells, immunoprecipitated with mouse anti-HA antibody (F7, Santa Cruz), eluted with HA peptide (Sigma, in 500 mM NaCl), and concentrated by the Centrifugal filter units (Millipore). GST-PH Gprk2 , GST-PH Gprk2RA , and GST-PH Gprk2FA proteins were expressed in bacteria and purified using the protocol employed for GST-Smo purification. The purified and concentrated GST-fusion proteins or epitope-tagged proteins were incubated with PI(4)P beads (Echelon Biosciences) with wash/binding buffer (10 mM HEPES, pH7.4; 0.25% NP-40; 150 mM NaCl), and subjected to western blot to detect PI(4)P bound proteins.

Protein-Lipid Overlay and PtdIns Lipid Pull-Down Assays
Immunostaining of Wing Imaginal Disc, Drosophila Embryo, and NIH3T3 Cell Cilia Wing discs from third instar larvae were dissected in PBS and then fixed with 4% formaldehyde in PBS for 20 min. After permeabilization with 1% PBST, discs were incubated with primary antibodies for 3 h and appropriate secondary antibodies for 1 h, and washed three times with PBST after each incubation. Affinity-purified secondary antibodies (Jackson ImmunoResearch) for multiple labeling were used. It was a challenge for disc staining with the mouse anti-PI(4)P antibody. We have adopted/modified a critical method for PI(4)P immunostaining from previous publications [46,65]. Discs were fixed in 4% formaldehyde in PBS and permeabilized in 1 M sucrose by freezing at -80°C for 1 h followed by thawing at room temperature. Then discs were washed with PBS and incubated with 50 mM NH 4 Cl for 15 min, followed by incubation with the anti-PI(4)P antibody at 4°C overnight. For Drosophila embryo primary antibody staining, stage 11 fly embryos with specific genotypes were dechorionated, fixed with Heptane solution, and immunostained with similar procedures. To examine mSmo ciliary localization, NIH3T3 cells were transfected with mSmo-GFP variants, treated with Shh or PI (4)P, and immunostained for mSmo localization in the cilium. Primary antibodies in this study were: mouse anti-Myc (9E10, Santa Cruz), anti-Flag (M2, Sigma), anti-SmoN (DSHB), anti-En (DSHB), and anti-PI(4)P (Z-P004, Echelon Biosciences); rabbit anti-β-Gal (Cappel), anti-GFP (Clontech), anti-Acetylated tubulin (Sigma), anti-PS1 [30], and rat anti-Ci (2A1, DSHB). Affinity-purified secondary antibodies (Jackson ImmunoResearch) for multiple labeling were used. Fluorescence signals were acquired on an Olympus confocal microscope and images processed with Olympus Fluoview Ver.1.7c. About 15 imaginal discs were screened and three to five disc images were taken for each genotype. The levels of Stt4 and Sac1 mRNA were monitored by Real-Time PCR when S2 cells were treated with 60% Hh-conditioned medium or 60% conditioned medium plus Hh cDNA transfection (to achieve the highest levels of Hh activity). No statistical differences detected. (B-C) Wing discs expressing Flag-Stt4 or GFP-Sac1 by MS1096-Gal4 were stained for Flag and GFP. A/P boundary was defined by Ci staining. There were no Flag and GFP staining differences between A and P compartments, indicating Hh does not regulate the stability of the protein.

Supporting Information
The underlying data of panel A can be found in S1 Data. RNAi efficiency in NIH3T3 cells was shown by western blot with the anti-Myc-antibody to detect the transfected Myc-Ptc1, and shRNA1 was used in this study. (B) NIH3T3 cells transfected with YFP-Ptc1 or GFP-Ptc2 were immunostained to show the expression of Acetylated (Ac)-tubulin (red; primary cilium), YFP (green; Ptc1), and GFP (green; Ptc2). Images in the inserts are enlarged views with shifted overlays to show the ciliary localization of Ptc1 or Ptc2. About 100 ciliated cells were counted for each set. Quantification of ciliary localization of Ptc1 or Ptc2 is shown in Fig 7G in the main text. The underlying data of panel A can be found in S1 Data. (TIF) S1 Methods. Additional methods. We provide additional information for the generation of various constructs, transgenic lines, and mutants. We also describe the procedures for GST fusion protein purification and in vitro kinase assay, luciferase assay, and cuticle preparation. (DOCX) S1 Table. RNAi screen for PITP involved in Smo regulation by PI(4)P. The results and phenotypes from knockdown of the indicated PITP by RNAi using different Gal4 lines are shown in the table. Cell culture assays for Smo stability and phosphorylation are also shown with the primers used for the synthesis of individual dsRNA. (DOCX)