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.
The Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis [1–3]. 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 [4–6]. 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) [14–16], 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 [21–23]. 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 [26–28]. 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 [36–38]. 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.
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 SmoPKA12, 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 SmoPKA12 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 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 SmoSD123, 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 PIP2 (PI (3,4) P2, PI (3,5) P2, PI (4,5) P2), and PIP3 (PI (3,4, 5) P3) [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 RNAi-mediated 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) P2-specific phosphatase to produce PI (4) P) enhanced, whereas IpgD (converts PI (4,5) P2 into PI (5) P) suppressed, the basal and Hh-induced ptc-luc activity (Figs 1F and S3A). As a control, Inp54pD281A 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. 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-PHOSBP 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-PHOSBP 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) P2 or PIP3 (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). 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-SmoWT 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 Myc-SmoΔC (Smo lacking the C-tail) did not interact with PI (4) P (Fig 3A, right column), suggesting that the C-tail of Smo is required for binding to PI (4) P. To identify the residues in the Smo C-tail interacting with PI (4) P, we used an in vivo approach to examine whether the activity of Smo is regulated by the PH domain expression. Membrane-tethered Smo C-terminal truncations by the myristoylation signal (Myr-SmoCT) possess the activity to induce ectopic dpp-lacZ expression [21]. We found that the membrane-tethered PH domain of OSBP (Myr-PHOSBP) increased the ectopic dpp-lacZ expression induced by Myr-Smo730-1035 but did not change dpp-lacZ expression induced by Myr-Smo764-1035 (compare Fig 3E to 3D and 3G to 3F), although Myr-PHOSBP itself had no effect on dpp-lacZ expression in the wing. This suggests that PHOSBP likely regulates Smo activity through aa 730–764. This domain of Smo contains one of the four positively charged arginine clusters, which are known to negatively regulate Smo activity by counteracting phosphorylation (Fig 3B) [20]. Surprisingly, using GST-Smo656-755 in the solid-phase lipid-binding assay, we found that SmoWT strongly interacted with PI (4) P; however, an Arg to Ala mutation (GST-SmoRA4) 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 Smo full-length (Myc-SmoRA4) and found that Myc-SmoRA4 lost both the ability to bind PI (4) P (Fig 3J) and the interaction with Cos2-Fu complex (Fig 3K). In addition, phosphorylation of SmoRA4 was no longer regulated by Hh and PI (4) P (Fig 3K). SmoRA4 also had no responsiveness to PI (4) P stimulation in the ptc-luc assay (S4E Fig). Using the fluorescence resonance energy transfer (FRET) assay to test C-terminal CFP/YFP dimerization [20,44], we found that SmoRA4 had a much lower FRET signal and was much less responsive to both Hh and PI (4) P stimulation compared to SmoWT (S4F Fig). RA4 mutation did not cause protein misfolding because there was no difference between SmoRA4 and SmoWT regarding the expression and subcellular distribution in S2 cells. Taken together, our findings suggest that SmoRA4 is inactive. Indeed, driven by the tubulinα promoter that expresses Smo at a level close to endogenous gene expression [17], the expression of SmoRA4 did not rescue ptc and en expression in smo mutant cells (Figs 3L, 3M and S4G). Furthermore, we found that overexpression of SmoRA4 by MS1096-Gal4 did not induce ectopic expression of dpp-lacZ, as noted with SmoWT (Fig 3N and 3O), and that an R>A mutation in the constitutively active form of Smo (SmoSD123RA4) had lower ptc-luc activity compared to SmoSD123 (S4E Fig) and was unable to induce en expression in ventral A compartment cells (Fig 3R, compared to 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 (SmoRE4), 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. The binding position of PI (4) P in Smo is very critical, because fusion of the PH domain from OSBP to either the third intracellular loop (SmoL3PH) or the C-tail (SmoPH) retained PI (4) P with Smo (S4A Fig) but compromised Smo activity in the wing (S4C and S4D Fig, compared to S4B Fig) and resulted in loss of responsiveness to PI (4) P stimulation (S4E Fig). 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 position-dependent manner. 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-SmoPKA12 (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 (Grpk2ΔC) abolished its ability to rescue en expression (Fig 4B), whereas replacing the PH domain in Gprk2 with the PH domain from OSBP (Gprk2-PHOSBP) 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, Gprk2ΔC, and Gprk2-PHOSBP, 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 Gprk2ΔC 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 Gprk2ΔC had no effect (Fig 4G and 4H). Similar to RFP-PHOSBP (Fig 2B and 2C), overexpression of PHGprk2 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′), whereas Gprk2ΔC 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′-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-PHOSBP rescued Smo phosphorylation inhibited by RNAi of Gprk2 3′-UTR but the expression of HA-Gprk2ΔC 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 (PHGprk2) interacted directly with PI (4) P; mutation of arginine (PHGprk2RA) or phenylalanine (PHGprk2FA) abolished this interaction (Fig 5F). Finally, similar to the PHOSBP interaction with Smo (Fig 2H and 2I), the PHGprk2 interaction with Myc-SmoWT 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. 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 transmembrane molecular transporter [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 Ptc-mediated 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-PtcWT), HA-tagged Ptc lacking its SSD domain (HA-PtcΔSSD), 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-PtcWT and HA-SSD strongly bound PI (4) P, whereas HA-PtcΔSSD 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 PIP2 or PIP3 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 PtcWT 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, PtcΔSSD 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. 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, PIP2 and PIP3 treatment had no effect on mSmo activity. In addition, the activity of the constitutively active form of mSmo (mSmoSD), which mimics mSmo phosphorylation by GRK2 and CK1 [30], was further increased by PI (4) P (Fig 7B). Consistently, Drosophila SmoSD123 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 mSmoWT (Fig 7C). However, R>A mutations in R3 and R4 arginine clusters (mSmoRA3 and mSmoRA4, respectively) attenuated the increased activity noted with PI (4) P, and mutations in both R3 and R4 (mSmoRA34) 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. mSmoWT was found in about 5% of cilia, and Shh treatment increased SmoWT accumulation in 75% of cilia (Fig 7D and 7E) [30]. Treatment with PI (4) P induced mSmoWT accumulation in 47% of cilia (Fig 7D and 7E), which was correlated with changes in mSmoWT phosphorylation (Fig 2F) and activity (Fig 7B) induced by PI (4) P. In contrast, mSmoRA34 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, mSmoRA34 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).
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 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. 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 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 [60–62]. 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). Binding of PI (4) P likely changes the conformation of Smo, leading to Smo phosphorylation by kinases. It should be noted that phosphomimetic Smo mutations (SmoSD123 and mSmoSD) are still regulated by PI (4) P (Figs 7B and S4E), suggesting that PI (4) P either promotes phosphorylation at other residues or has additional role (s) in activating Smo. 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 wild-type 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 SmoSD123 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) P was accumulated in wing discs (S2A Fig), likely due to the high levels of Hh signaling activity induced by the active forms of Smo and Ci.
Generation of the Myc-SmoWT, Myc-SmoΔC, Myr-Smo730-1035, and Myr-Smo764-1035 constructs and transgenes was previously described [21]. Myc-SmoPH and Myc-SmoL3PH were constructed by fusing one copy of the OSBP PH domain (aa 17–123) to the Smo C-terminus and two copies of the OSBP PH domain after aa 451 of Smo intracellular loop 3, respectively. Generation of the Myc-SmoSD123 and Myc-SmoRA1234 was previously described [15,20]. Myc-SmoSD123RA4 was generated by the combination of SD123 and RA4. Myc-tagged SmoRA1, SmoRA2, SmoRA3, SmoRA4, SmoRA12, and SmoRA34 were generated by PCR. To construct tub-SmoRA4, the previously described tubulinα promoter [17] was inserted upstream to the SmoRA4 sequence. Transgenic lines were generated using the VK5 attP locus to ensure Smo protein expression at the same levels without positional effects. Genotypes for examining the activity of Smo transgene in smo clones include: yw hsp-flp/+ or Y, smo3 FRT40/hs-GFP FRT40, and tub-SmoRA4/+. RFP-PHOSBP and RFP-PHGprk2 were constructed by an in-frame fusion of two copies of the PH domain from OSBP or Gprk2 at the RFP C-terminus in the attB-UAST backbone [41]. RFP-PHOSBP and RFP-PHGprk2 transgenes were generated by insertion at the VK5 attP locus. As a control, the UAS-RFP transgenic line was generated with the same approach. Flag-Gprk2WT and Flag-Gprk2KM constructs and transgenes have been described [18]. Flag-tagged Gprk2ΔC (containing Gprk2 aa 1–666) and Gprk2-PHOSBP (aa 667–714 replaced by the PH domain of OSBP) were inserted in-frame in the Flag-UAST vector, and their transgenic lines were generated using standard P-element-mediated transformation. Multiple independent transgenes were generated, and those on the second chromosomes were used for rescue experiments. Gprk2 mutant clones were generated with yw 122 and FRT82 Gprk2/FRT82 hs-Myc-GFP. ptc mutant clones were generated with yw hsp-flp/+ or Y and ptc[wII] FRT42D/hs-GFP FRT42D. Inp54p (from Saccharomyces cerevisiae, Addgene 20155) and IpgD (from Shigella flexneri, a gift from Dr. Frederique Gaits-Iacovoni) were subcloned into HA-UAST backbones. A phosphatase-dead version of Inp54p with a D281A mutation (Inp54pD281A) was generated by site-directed mutagenesis. GST-SmoWT containing aa 656–755 has been described [15]. GST-tagged SmoRA12, SmoRA34, and SmoRA4 were generated by PCR on the backbone of GST-SmoWT. GST-PHGprk2, GST-PHGprk2RA, and GST-PHGprk2FA were constructed by the same approach. HA-PtcWT, HA-PtcΔSSD, and HA-SSD were generated by PCR with Ptc or its fragments inserted into HA-UAST backbones, and transgenic lines were generated using the VK5 attP locus. His-SSD was generated by in-frame fusion of Ptc SSD domain (aa421-589) to pET30a backbone. Myc-tagged mSmoWT, mSmoRA mutants, and mSmoSD (previous mSmoSD0-5) have been described [20,30]. GFP-tagged mSmoWT and mSmoRA34 were generated by subcloning each cDNA into pEGFP-N1 backbone. Flag-tagged Stt4 (Flag-Stt4) was generated by combining seven RT-PCR fragments with unique cloning sites into the SK+ backbone and, finally, subcloning into Flag-UAST backbone. HA-tagged Sac1 (HA-Sac1) was constructed by inserting the RT-PCR fragment into the HA-UAST backbone. pGE-mPtch1-Myc and pGE-mPtch2-Myc were generated by subclone of the open reading frame from pEGFP-mPtch1 and pEGFP-mPtch2 (Gift from Dr. Chi-Chung Hui), respectively. MS1096-Gal4, ap-Gal4, C765-Gal4, and Gprk2 deletion mutants have been described [17,18]. Stt4 RNAi lines (v15993 and v105614), Sac1 RNAi lines (v44376, v37216, and #56013), Ptc RNAi line (#28795), Hh RNAi line (v1402), GFP-Sac1 (#57356 and #57357), and prd-Gal4 (#1947) were obtained from VDRC or Bloomington Stock Center (BSC). Generation of Gprk2 RNAi lines have been described [18]. The RNAi lines that targeted each PITP were obtained from either VDRC or BSC (S1 Table). PtdIns lipid extraction and quantification by mass spectrometry were carried out as previously described [43]. Briefly, 3 × 107 S2 cells (or 4 × 106 NIH3T3 cells) from a 100 mm dish were harvested and washed once with ice-cold PBS and suspended in 340 μL H2O 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 CHCl3 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) P2 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. GST-Smo fusion proteins expressed in bacteria were pooled by GST beads (GE Healthcare), then eluted with elution buffer (10mM Glutathione pH 8. 0 in 50 mM Tris) at 4°C overnight. Myc-tagged Smo proteins expressed in S2 cells were immunoprecipitated with anti-Myc antibody combined with beads of protein A ultralink resin, followed by two sequential elutions with Myc peptide (Roche, 100mM KCl, 20% glycerol, 20 mM HEPES KOH, pH 7. 9,0. 2 mM EDTA, 0. 1% NP-40,5 mM DTT, and 0. 5 mM PMSF). The eluted purified GST-fusion proteins and Myc-tagged Smo proteins were concentrated by the Centrifugal filter units (Millipore) and incubated with lipid-dotted strips according to manufacturer’s instruction (Echelon Biosciences), followed by western blot with the anti-GST (Santa Cruz), anti-Myc (Santa Cruz), or anti-His (Millipore) antibodies. 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-PHGprk2, GST-PHGprk2RA, and GST-PHGprk2FA 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. 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 NH4Cl 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 Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis. A critical step in Hh signal transduction is the inhibition of Smoothened (Smo), an atypical G protein-coupled receptor (GPCR), by the Hh receptor Patched (Ptc). It is a longstanding question how Ptc inhibits Smo and how Hh promotes Smo phosphorylation and activation. It is unlikely that Ptc inhibits Smo by direct interaction. Here, we uncover that phosphatidylinositol 4-phosphate (PI (4) P), a specific phospholipid, directly interacts with Smo through an arginine motif in the Smo C-terminal tail and promotes Smo phosphorylation, activation, and ciliary localization. Ptc also interacts with PI (4) P, which is inhibited by Hh stimulation, indicating that Hh triggers the release of PI (4) P from Ptc. We further uncover that Hh stimulation induces the overall production of PI (4) P, likely by regulating PI (4) P kinase and phosphatase. Finally, in addition to the direct role in regulating Smo phosphorylation, G protein-coupled receptor kinase 2 (Gprk2) facilitates PI (4) P interaction with Smo. This study suggest that PI (4) P acts as a special small molecule shuttling between Ptc and Smo to modulate Hh responses.
phosphorylation
rna interference
enzyme-linked immunoassays
chemical compounds
organic compounds
immunoprecipitation
basic amino acids
amino acids
epigenetics
immunologic techniques
cellular structures and organelles
research and analysis methods
lipids
genetic interference
proteins
gene expression
immunoassays
chemistry
precipitation techniques
hedgehog signaling
biochemistry
signal transduction
rna
arginine
cell biology
post-translational modification
nucleic acids
organic chemistry
cilia
genetics
biology and life sciences
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cell signaling
PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail