Ezrin phosphorylation depends on the kinase activity of Kit mutants. (A) Ezrin is tyrosine-phosphorylated in 606 and 931 HS2 cells. Cells were treated or not for 4 hours with Kit inhibitors: PP1 (5 μM) and IM (1 μM). Ezrin was immunopreciptated from cell extracts treated with 0.1 mM pervanadate for 10 minutes. Immunoprecipitates were analyzed by Western blot using antiphosphotyrosine antibodies (PY) followed by an anti-ezrin antibody. Ig corresponds to a rabbit immunoglobulin immunoprecipitation. Inputs corresponded to 1:1000 immunoprecipitated cell extracts from 606 and 931 HS2 cells. (B) Tyrosine phosphorylation of ezrin by Kit^D814Y in COS-7 cells. COS-7 cells were transiently transfected with expression vectors coding for Ez^WT-VSVG and/or Kit mutants. Ezrin was immunoprecipitated from cell extracts with the anti-VSVG antibody, and the immunoprecipitates were analyzed by Western blot using antiphosphotyrosine (4G10), anti-kit, and anti-VSVG antibodies. The phosphorylated proteins are indicated by arrowheads. VSVG-Ezrin and Kit expression were controlled in WCEs from transfected cells with the indicated antibodies. (C) Sensitivity of the ezrin/Kit interaction to PP1, PP2, and IM. COS-7 cells were transiently transfected with expression vectors coding for Ez^WT-VSVG and/or Kit^D814Y and treated or not for 1 hour with PP1 (5 μM), PP2 (5 μM), or IM (1 μM). Cell extracts were treated with pervanadate (0.1 mM for 10 minutes) and subjected to immunoprecipitation with anti-ezrin antibody. The immunoprecipitates were analyzed by Western blot using antiphosphotyrosine antibodies (P-Y) and anti-VSVG antibodies. Ezrin phosphorylation was detected on Western blot with anti-PY (P-Y) and anti-VSVG antibodies. The expression and phosphorylation of Kit were controlled on WCEs with the anti-PY and anti-Kit antibodies. (D) Tyrosine phosphorylation of ezrin by the oncogenic Flt3^D835Y or TEL-Jak2 kinases. COS-7 cells were transfected with expression vectors coding for Kit^D814Y or Flt3^D835Y or TEL-Jak2 and VSVG-ezrin. Ezrin was immunoprecipitated from transfected COS-7 cell extracts with the anti-VSVG antibody, and the immunoprecipitates were analyzed by Western blot using antiphosphotyrosine (P-Y) and anti-VSVG antibodies. The expression of the transfected proteins in WCEs was confirmed by Western blotting using the antibodies indicated on the right of the blots. Vertical lines have been inserted to indicate repositioned gel lanes. (E) Tyrosine phosphorylation of ezrin by wild-type Kit. COS-7 cells were transfected with expression vectors coding for Kit^WT and VSVG-ezrin in the presence or in the absence of SCF. Ezrin was immunoprecipitated from transfected COS-7 cell extracts with the anti-VSVG antibody, and the immunoprecipitates were analyzed by Western blot using antiphosphotyrosine (P-Y) and anti-VSVG antibodies. The expression and phosphorylation of Kit in WCEs were confirmed by Western blotting using the anti–P-Y and anti-Kit antibodies.