Through the action of its membrane bound type I receptor, TGF-ß elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-ß and its related factors. Here we show that TGF-ß induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and GSK3 is responsible for phosphorylation at one of these sites, namely Ser204. Alanine substitution at Ser204 and/or the neighboring Ser208, the priming site for GSK3 in vivo activity, strengthened the affinity of Smad3 to CBP, suggesting that the linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-ß.

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