Transforming growth factor-{beta}-inducible phosphorylation of Smad3.
Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta 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, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta 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-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.
Wang
Guannan
G
Center for Advanced Biotechnology and Medicine.
Matsuura
Isao
I
He
Dongming
D
Liu
Fang
F
D000818:Animals; D045744:Cell Line, Tumor; D005347:Fibroblasts / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D051379:Mice; D008907:Mink; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051900:Smad3 Protein / Q000502:physiology*; D013912:Threonine / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D005419:Flavonoids; D008024:Ligands; D010880:Piperidines; D047428:Protein Kinase Inhibitors; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D013912:Threonine; C077990:alvocidib