PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail
Fig 5
PH domain is required for Gprk2 to promote Smo phosphorylation.
(A) An in vitro kinase assay using the GST-mSmo (mouse Smo aa 608–670) fusion protein and the indicated Flag-tagged Gprk2 proteins purified from S2 cells. (B) NIH3T3 cells were transfected with the indicated constructs and immunoprecipitated with the anti-Myc antibody, followed by western blot with the anti-PS1 antibody to detect mSmo phosphorylation. Of note, expressing Gprk2ΔC induces mSmo phosphorylation at a level comparable to expressing Gprk2WT. (C) S2 cells were transfected with Myc-SmoWT and GFP, treated with the indicated dsRNA in combination with either PI(4)P or carrier control, followed by immunoprecipitation using the anti-Myc antibody and western blot analysis using the anti-SmoP and anti-Myc antibodies. Myc-Smo normalization is described in S1 Methods. GFP served as transfection control. (D) S2 cells were transfected with Myc-SmoWT and GFP, treated with either dsRNA targeting the coding region of Gprk2 or dsRNA targeting the 3′-UTR of Gprk2 in combination with transient transfection of HA-Gprk2, HA-Gprk2ΔC, or HA-Gprk2-PHOSBP. Cell extracts were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis using the anti-SmoP and anti-Myc antibodies. Myc-Smo normalization is described in S1 Methods. GFP served as transfection control. (E) A PI(4)P beads pull-down assay examined binding of HA-Gprk2WT or HA-Gprk2ΔC purified from S2 cells transfected with each construct. (F) A PI(4)P beads pull-down assay examined bound GST-Gprk2 proteins purified from bacteria. (G–H) S2 cells were transfected with Myc-SmoWT and HA-PHGprk2 followed by treatment with Hh-conditioned medium (30% or 60%) or PI(4)P (5 μM or 10 μM). Immunoprecipitated cell extracts using the anti-Myc antibody was followed by western blot analysis with either the anti-Myc or anti-HA antibody to detect Smo bound PHGprk2. Lysates blotted with the anti-HA antibody served as a transfection and loading control.