Blockade of TGFbeta3 up-regulation of p27Kip1 and p21Cip1 by expression of RasN17 in epithelial cells.
Our previous data demonstrated that Ras activation is necessary and sufficient for transforming growth factor beta (TGFbeta)-mediated Erk1 activation, and is partially required for the inhibition of cyclin-dependent kinase 2 (Cdk2) activity, cyclin A expression and DNA synthesis by TGFbeta (KM Mulder and SL Morris, J. Biol. Chem., 267: 5029-5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270: 7117-7124, 1995; and MT Hartsough et al., J. Biol. Chem., 271: 22368-22375, 1996). Here, we examined the kinetics and role of Ras in TGFbeta3-mediated effects on specific G1 cell cycle components in TGFbeta-sensitive (4-1) and TGFbeta-resistant (4-6) intestinal epithelial cells (IEC's). Our results indicate that inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) completely abrogated the ability of TGFbeta3 to up-regulate both CKI's. In contrast, the ability of TGFbeta3 to up-regulate p27Kip1 and p21Cip1 was maintained in ZnCl2-treated control cells. Inactivation of Ras also completely blocked the rapid TGFbeta-mediated increase in new synthesis of p27Kip1 protein. Moreover, up-regulation of p21Cip1 protein levels and new synthesis of p27Kip1, as well as the association of these CKI's with Cdk2, preceded the decrease in Cdk2 activity by TGFbeta. Collectively, our results suggest that p21Cip1 and p27Kip1 are upstream effectors of the TGFbeta-mediated inhibition of Cdk2 activity in IEC 4-1 cells, and demonstrate that Ras activation is obligatory for TGFbeta-mediated up-regulation of these CKIs in untransformed epithelial cells.
Yue
J
J
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Buard
A
A
Mulder
K M
KM
D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation; D018631:ras Proteins / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D011494:Protein Kinases; C102333:histone H1 kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D018631:ras Proteins