T-BETA-RI PHOSPHORYLATION OF SMAD2 ON SER(465) AND SER(467) IS REQUIRED FOR SMAD2-SMAD4 COMPLEX-FORMATION AND SIGNALING

Mothers against Dpp-related or Smad proteins are essential components of serine/threonine kinase receptor signaling pathways that are regula ted by phosphorylation. Recently, it was demonstrated that Smad2 inter acts transiently with and is a direct substrate of the transforming gr owth factor-beta (TGF-beta) type I receptor, T beta RI. Phosphorylatio n sites on Smad2 were localized to a carboxyl-terminal fragment contai ning three serine residues at positions 464, 465, and 467. In this rep ort, we show that T beta RI specifically phosphorylates Smad2 on serin es 465 and 467. Serine 464 is not a site of phosphorylation, but is im portant for efficient phosphorylation of Smad2. Phosphorylation at bot h sites is required to mediate association of Smad2 with Smad4 in mamm alian cells, while in yeast, Smad2 interacts directly with Smad4 and d oes not require phosphorylation. Mutation of either serine residue 465 or 467 prevents dissociation of Smad2 from activated T beta RI and bl ocks TGF-beta-dependent signaling and Smad2 transcriptional activity. These results indicate that receptor-dependent phosphorylation of Smad 2 on serines 465 and 467 is required in mammalian cells to permit asso ciation with Smad4 and to propagate TGF-beta signals.