Tec and Dok phosphorylation depends on PI3-K activity.
F36P cells were incubated with different concentrations of the PI3-K inhibitors wortmannin (WM; 20 minutes; A) or LY294002 (LY; 60 minutes; B) at 37°C, after which the cells were incubated with SCF for 5 minutes. (A) Tec immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine (top panel) and anti-Tec (lower panel). WM clearly inhibits Tec and Dok-1 phosphorylation. (B) Lysates were incubated with anti-Tec and anti-Dok-1. Tec immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine and anti-Tec; Dok precipitates were analyzed with antiphosphotyrosine. LY clearly inhibits Tec and Dok-1 phosphorylation. Whole cell lysates (1 × 106 cells) were also analyzed with antiphospho-PKB and antiphospho-ERK as control for the specificity of LY. LY inhibits PKB phosphorylation but not ERK phosphorylation. (C) The 293 cells were transfected with expression vectors encoding Tec, Dok-1, or Δp85, or with empty vector (−). Tec and Dok-1 immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine (top panels) and anti-Tec (lower left panel). Tyrosine phosphorylation of Tec and Dok-1 can be blocked by coexpression of dominant negative PI3-K (Δp85).
