FIGURE 5.

GSK3 is the kinase for TGF-β-induced phosphorylation of Ser²⁰⁴. A, LiCl selectively inhibits Ser²⁰⁴ phosphorylation. Mv1Lu cells were pretreated with 120 mm NaCl (control) or LiCl for 1 h before addition of TGF-β. The Smad3 linker phosphorylation was analyzed by phosphospecific antibodies. B, TGF-β-induced phosphorylation of Ser²⁰⁴ is selectively inhibited in GSK3β^–/– MEF. GSK3β^–/– MEF and the wild type (WT) littermate control MEF were treated with TGF-β for 1 h. Phosphorylation levels of the three linker sites in Smad3 were examined. C, GSK3β can phosphorylate the Ser²⁰⁴ site in vitro. 100 ng of recombinant GSK3β was used to phosphorylate 0.5 μm GST-Smad3 in a nonradioactive reaction. The reaction products were analyzed for Ser²⁰⁴ phosphorylation by immunoblotting with the Ser(P)²⁰⁴ antibody. D, Ser²⁰⁸ is the priming site for Ser²⁰⁴ phosphorylation. 293 cells were transfected with wild type Smad3 or the S208A mutant, together with the activated TβRI. Cell lysates were analyzed for Ser²⁰⁴ phosphorylation by the phosphospecific antibody.