Cloning and expression of a rat Smad1: regulation by TGFbeta and modulation by the Ras/MEK pathway.
A new family of signaling intermediates for TGFbeta superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFbeta superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFbeta induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFbeta receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFbeta to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFbeta receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFbeta-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFbeta can regulate Smadl and that the Ras and MEK signaling components are partially required for the ability of TGFbeta to regulate Smad1.
Yue
J
J
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Hartsough
M T
MT
Frey
R S
RS
Frielle
T
T
Mulder
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004330:Drosophila; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D048369:MAP Kinase Kinase 1; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D009928:Organ Specificity; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism*