Transforming Growth Factor-β-inducible Phosphorylation of Smad3

Smad proteins transduce the transforming growth factor-β (TGF-β) signal at the cell surface into gene regulation in the nucleus. Upon TGF-β treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-β receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser208, Ser204, and Thr179 are phosphorylated in response to TGF-β. The linker phosphorylation peaks at 1 h after TGF-β treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-β receptor is necessary for the TGF-β-induced linker phosphorylation. Although the TGF-β receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-β-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-β-inducible Ser204 phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-β-induced phosphorylation of Thr179 and Ser208, suggesting that the CDK family is responsible for phosphorylation of Thr179 and Ser208 in response to TGF-β. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-β/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-β-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.