Figure 8.

Erk activation by TGF-β is inhibited by inactive ShcA mutants and ShcA downregulation. 3T3-Swiss cells were infected with adenoviruses expressing GFP (control), HA-tagged p52^ShcA lacking the PTB domain (p52^ShcAΔPTB), or HA-tagged p52^ShcA lacking the SH2 domain (p52^ShcAΔSH2). Cells were then stimulated with TGF-β for the indicated times and lysed. (A) TGF-β-induced tyrosine phosphorylation of endogenous ShcA is attenuated by expression of p52^ShcA truncations. Endogenous ShcA was immunoprecipitated and subjected to phosphotyrosine Western analysis (above). HA immunoblotting of this membrane confirmed expression of p52^ShcA truncations and equivalent precipitation efficiency (below). (B) TGF-β-induced Grb2 association with endogenous ShcA is attenuated by p52^ShcA truncations. The membrane in (A) was subjected to Grb2 Western analysis. (C) TGF-β-induced Raf phosphorylation is attenuated by p52^ShcA truncations. Cell lysates from the experiment shown in (A) were subjected to phosphoRaf Western blot. (D) TGF-β-induced Erk phosphorylation is attenuated by p52^ShcA truncations. The membrane shown in (C) was subjected to phosphoErk1/2 Western blot (above). The membrane was reprobed for GAPDH to confirm equivalent loading (below). (E) TGF-β-induced Erk activation is attenuated by ShcA silencing and restored by ectopic expression of wild-type, but not mutant p52^ShcA. 3T3-Swiss cells were exposed to an siRNA targeted to the common region of ShcA together with adenoviruses encoding either green fluorescent protein (GFP), wild-type p52^ShcA, or p52^ShcA in which the Grb2 binding sites are mutated. Both p52^ShcA constructs incorporate a silent mutation in the siRNA target site. PhosphoErk immunoblot of cells stimulated with TGF-β for the indicated times (above). ShcA immunoblot confirming partial downregulation and virus-mediated expression (below).