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.