PI(4)P promotes Smo phosphorylation.
(A) A wing disc expressing UAS-RFP by MS1096-Gal4 was stained for Smo. The expression of RFP has no effect on Smo accumulation. (B–C) Wing discs expressing RFP-PHOSBP by MS1096-Gal4 were stained for PI(4)P and Smo. Arrows indicate accumulation of PI(4)P and Smo by PHOSBP expression. (D) S2 cells were transfected with Myc-SmoWT and GFP followed by the treatment with the indicated Ptdlns lipids, in combination with either Hh-conditioned medium or control medium. Cell extracts were immunoprecipitated with the anti-Myc antibody and analyzed by western blot using the anti-SmoP or anti-Myc antibodies. GFP served as a transfection and loading control. (E) S2 cells were transfected with Myc-SmoWT and GFP, followed by the treatment of PI(4)P and Hh-conditioned medium. Immunoprecipitation of cell extracts with the anti-Myc antibody was followed by western blot analysis using the anti-SmoP and anti-Myc antibodies. Myc-Smo was normalized by the method described in Methods. GFP served as a transfection control. (F) NIH3T3 cells were transfected with mSmo-Myc and treated with Shh or the indicated PIPs, followed by western blot analysis using the anti-PS1 antibody to examine mSmo phosphorylation. (G) An in vitro kinase assay is shown using the purified GST-SmoWT protein containing aa 656–755, the commercial PKA and CK1 kinases, and PI(4)P, PI(4,5)P2, PI(3,4,5)P3 (10 μM each). GST was used as a control. (H–I) S2 cells were transfected with Myc-SmoWT and HA-PHOSBP, followed by treatment with PI(4)P (5 μM or 10 μM) or Hh-conditioned medium (30% or 60%). Immunoprecipitation of cell extracts with the anti-Myc antibody was followed by western blot with either the anti-Myc or anti-HA antibody to detect Smo-bound PHOSBP. Lysates blotted with the anti-HA antibody served as loading control.