FIGURE 2.

The C-tail phosphorylation by the TGF-β receptor is necessary for TGF-β-induced phosphorylation of the Smad3 linker sites. A, TβRI is required for TGF-β-induced Smad3 linker phosphorylation. Mv1Lu-derived TβRI-deficient L17 cells were transfected with empty vector or TβRI. Cells were then treated with or without 300 pm TGF-β for 1 h. Phosphorylation of Ser²⁰⁸, Ser²⁰⁴, and Thr¹⁷⁹ were analyzed by immunoblots. A representative experiment is shown. The averages of four independent experiments were plotted. The error bars indicate standard deviation. B, the Smad3 C-tail phosphorylation is necessary for TGF-β-induced linker phosphorylation. L17 cells were cotransfected with TβRI along with either wild type (WT) Smad3 or the C-tail 3A mutant. Cells were then treated with TGF-β for 1 h or with EGF for 15 min for maximal induction. Phosphorylation of Ser²⁰⁸, Ser²⁰⁴, and Thr¹⁷⁹ were then analyzed by immunoblots. The bar graphs represent the averages of four independent experiments. The error bars indicate standard deviation. C, mutation of the Smad3 linker phosphorylation sites has essentially no effect on C-tail phosphorylation. L17 cells were cotransfected with TβRI along with either the wild type Smad3 or the linker phosphorylation mutant T179V/S204A/S208A. Cells were then treated with TGF-β for 1 h. Phosphorylation of the C-tail was examined by immunoblot.