title: Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells.
abstract: Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-beta signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-beta receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-beta treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-beta in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-beta signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-beta receptor complex is an essential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter.
authors.affiliation: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Sun
authors.forename: S N
authors.initials: SN
authors.lastname: Constantinescu
authors.forename: E
authors.initials: E
authors.lastname: Karam
authors.forename: R A
authors.initials: RA
authors.lastname: Weinberg
authors.forename: H F
authors.initials: HF
authors.lastname: Lodish
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008907:Mink; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D051900:Smad3 Protein; D015534:Trans-Activators; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta