title: Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein.
abstract: Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 27710, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Yingling
authors.forename: M B
authors.initials: MB
authors.lastname: Datto
authors.forename: C
authors.initials: C
authors.lastname: Wong
authors.forename: J P
authors.initials: JP
authors.lastname: Frederick
authors.forename: N T
authors.initials: NT
authors.lastname: Liberati
authors.forename: X F
authors.initials: XF
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D004247:DNA