Abstract
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.
Highlights
TGF- binds directly to the type II receptor, which leads to the recruitment of the type I receptor into the ligand-receptor complex
Upon TGF- treatment, Smad2 and Smad3 are phosphorylated by the TGF- type I receptor at the SSXS motif in their C-tails [11, 12], form complexes with Smad4, together accumulate in the nucleus to regulate transcription of target genes [2,3,4,5,6,7,8]
We further show that mutation of the linker phosphorylation sites increases the ability of Smad3 to activate a TGF-/Smad target gene and to inhibit cell proliferation
Summary
These observations suggest that TGF--induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity. We show in this report that TGF- treatment induces rapid phosphorylation of Ser208, Ser204, and Thr179 in the Smad3 linker region. We further show that mutation of the linker phosphorylation sites increases the ability of Smad3 to activate a TGF-/Smad target gene and to inhibit cell proliferation.
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