TRANSFORMING-GROWTH-FACTOR BETA-INDUCED PHOSPHORYLATION OF SMAD3 IS REQUIRED FOR GROWTH-INHIBITION AND TRANSCRIPTIONAL INDUCTION IN EPITHELIAL-CELLS

Drosophila Mad proteins are intracellular signal transducers of decape ntaplegic (dpp), the Drosophila transforming growth factor beta (TGF-b eta)/bone morphogenic protein (BMP) homolog. Studies in which the mamm alian Smad homologs were transiently overexpressed in cultured cells h ave implicated Smad2 in TGF-beta signaling, but the physiological rele vance of the Smad3 protein in signaling by TGF-beta receptors has not been established, Here me stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highl y efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-beta treatment Smad3 becomes rapidly phosphorylate d at the SSVS motif at its very C terminus. Either attachment of an ep itope tag to the C terminus or replacement of these three serine resid ues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; the se proteins act in a dominant-negative fashion to block the antiprolif erative effect of TGF-beta in mink lung epithelial cells. A Smad3 prot ein in which the three C-terminal serines have been replaced by aspart ic acids is also a dominant inhibitor of TGF-beta signaling, but can a ctivate plasminogen activator inhibitor 1 (PAI-1) transcription in a l igand-independent fashion when its nuclear localization is forced by t ransient overexpression, Phosphorylation of the three C-terminal serin e residues of Smad3 by an activated TGF-beta receptor complex is an es sential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter.