FIGURE 6.

The CDK family members mediate the TGF-β-induced linker phosphorylation. A, flavopiridol, a pan-CDK and GSK3 inhibitor, inhibits TGF-β-induced linker phosphorylation. Mv1Lu cells were treated with or without 250 nm flavopiridol for 8 h. Cells were then incubated with TGF-β for 1 h. Phosphorylation of Smad3 in the linker region was analyzed. The Smad3 C-tail phosphorylation was also analyzed as a control. B, both CDK2 and CDK4 can phosphorylate Thr¹⁷⁹ and Ser²⁰⁸ in vitro. CDK2 or CDK4 immunoprecipitated from 240 μg of Mv1Lu cell lysates was used to phosphorylate 1 μm GST-Smad3 in a nonradioactive reaction. IgG was also used to immunoprecipitate the lysates and the immunoprecipitates were used in the control kinase reaction (in the – lane). The reaction products were analyzed by immunoblotting with Thr(P)¹⁷⁹ or Ser(P)²⁰⁸ antibodies.