FIGURE 7.

p66^shc impairs IGF-I-stimulated Src kinase activation through polyproline-SH3 domain and SH2-phosphotyrosine interactions in living cells. A–C, quiescent LacZ, WT p66^shc (p66^shc WT)-, and p66^shc P3A-overexpressing cells were preincubated with or without p189 or p136 for 2 h before stimulation with IGF-I (100 ng/ml) for the indicated times. The activation of Src kinase was measured by probing with an anti-phospho-Tyr⁴¹⁹ Src antibody. The blots were reprobed with an anti-Src antibody to control for loading differences. p < 0.05 (*) and p < 0.01 (**) indicate the significant differences between two different treatments or the same treatment in two cell types. D, pSMC were transduced with pLenti-p66^shc WT-HA (p66^shc WT) or pLenti-p66^shc ΔSH2-HA (p66^shc ΔSH2) vector. Both cell types were serum-starved for 16–18 h and analyzed for HA and p66^shc protein expression. Cell lysates were immunoblotted with anti-HA and p66^shc antibodies. The arrows denote the 66-kDa and SH2-deleted p66^shc forms. E, quiescent LacZ, p66^shc WT, and p66^shc ΔSH2 cells were stimulated with IGF-I (100 ng/ml) for the indicated times. Cell lysates were immunoblotted with an anti-phospho-Tyr⁴¹⁹ Src antibody. To control for loading, the blot was reprobed with an anti-Src antibody. p < 0.01 (**) and p < 0.05 (*) indicate the significant differences between two cell types. The figure is representative of at least three independent experiments. IB, immunoblot; Ctrl, control.