Through the action of its membrane-bound type I receptor, transforming growth factor-␤ (TGF-␤) 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-␤ and its related factors. Here, we show that TGF-␤ induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREBbinding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-␤.

Signaling of transforming growth factor beta (TGF-beta) is mediated through a heteromeric complex of two types of transmembrane receptors and downstream intracellular proteins known as Smads. Alterations of TGF-beta signaling underlie various forms of human cancer and developmental diseases. Human genetic studies have revealed both point mutations and deletions of Smad2 or Smad4 in several types of cancers. However, the role of Smad3 in tumorigenesis is not clear. Recent data indicate that Smad3 also functions as a tumor suppressor by inhibiting cell proliferation and promoting apoptosis. In addition, Smad3 is essential for TGF-beta-mediated immune suppression, and it plays an important role in regulating transcriptional responses that are favorable to metastasis. Therefore, through regulating different transcriptional responses, Smad3 functions as both a negative and positive regulator of carcinogenesis depending on cell type and clinical stage of the tumor.