Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling.
The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.
Ueki
Nobuhide
N
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-5222, USA.
Hayman
Michael J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D017930:Genes, Reporter; D008907:Mink; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta