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Fig. 7. EGF signaling to p120 Y228 in A431 cells is independent of Src. (A) Validation of SU6656 activity. Phosphorylation of p120 by Src was monitored by in vitro kinase assay in the presence of increasing concentrations of the Src-family inhibitor SU6656. 32P-labeled samples were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography (15-minute exposure). As a control for sample loading, p120 was subsequently quantitated by western blotting with mAb-pp120 (5-second exposure). (B) EGF-induced p120 phosphorylation at Y228 in A431 cells is not blocked by SU6656. A431 cells were serum starved for 18 hours, then treated for 1 hour with DMSO alone or DMSO containing the indicated concentrations of SU6656. Cells were then incubated for 5 minutes with or without EGF (100 ng/ml), and lysed in RIPA containing pervanadate. Total p120 (p120) and Y228-phosphorylated p120 (pY228) levels were quantitated by western blotting. EGFR phosphorylation was determined by western blotting EGFR immunoprecipitates with the phosphotyrosine-specific mAb PY20 (EGFR, PY20) or with an EGFR-specific mAb (EGFR). (C) Src-independent EGF-induced p120 phosphorylation in HER-14 cells. HER-14 cells are murine Swiss 3T3 fibroblasts stably transfected with a human EGFR construct. The cells were serum starved for 12.5 hours in the presence of 4 µg/ml each insulin and transferrin to improve viability, then incubated for one hour with DMSO alone or with DMSO containing 2 µM SU6656. Cells were then treated for 5 minutes with or without EGF (100 ng/ml) prior to lysis in RIPA including pervanadate. p120 immunoprecipitates generated with mAb 15D2 were divided, and then western blotted with mAb pY228 or mAb pp120. (D) EGF induces p120 Y228 phosphorylation in the absence of Src kinase activity. SYF cells are murine embryonic fibroblasts derived from mice that are null for Src, Yes and Fyn. Other Src-family kinases have not been detected in these cells. Polyclonal EGFR-expressing cell lines were generated from SYF cells expressing either c-Src (+c-Src) or kinase dead c-Src (kd-Src). Cells were serum starved for 24 hours, EGF stimulated (100 ng/ml) and analyzed as described in B for p120 phosphorylation at Y228.
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