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.
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We thank the Shared Resource Facilities of the University of Kentucky Markey Cancer Center (supported by NCI grant P30 CA177558). This study was supported by National Institutes of Health grant R01 GM079684 to JJ.
Jiang, Kai; Liu, Yajuan; Fan, Junkai; Zhang, Jie; Li, Xiang-An; Evers, B. Mark; Zhu, Haining; and Jia, Jianhang, "PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened Through Interaction with Its C-terminal Tail" (2016). Markey Cancer Center Faculty Publications. 70.
S1 Data. Numerical data used in preparation of Figs 1C–1F, 7B, 7C, 7E–7G, S3A–S3D, S4E, S4F, S6A and S7A.
journal.pbio.1002375.s002.TIF (1881 kB)
S1 Fig. Inactivation of Stt4 and Sac1 by RNAi modifies the Hh phenotypes in the wing and larva.
journal.pbio.1002375.s003.TIF (3720 kB)
S2 Fig. The overexpression of constitutively active forms of Ci or Smo induces the accumulation of PI(4)P in vivo.
journal.pbio.1002375.s004.TIF (465 kB)
S3 Fig. PI(4)P treatment elevates Hh signaling activity monitored by the ptc-luc reporter.
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S4 Fig. PH domain fusion at the C-tail or the third intracellular loop compromised Smo activity.
journal.pbio.1002375.s006.TIF (2372 kB)
S5 Fig. Gprk2 regulates the levels of PI(4)P in wing disc.
journal.pbio.1002375.s007.TIF (1872 kB)
S6 Fig. Hh does not regulate the transcription and protein stability of Stt4 and Sac1.
journal.pbio.1002375.s008.TIF (975 kB)
S7 Fig. Shh activity promotes the production of PI(4)P in NIH3T3 cells.
journal.pbio.1002375.s009.DOCX (48 kB)
S1 Methods. Additional methods.
journal.pbio.1002375.s010.DOCX (21 kB)
S1 Table. RNAi screen for PITP involved in Smo regulation by PI(4)P.