Blockade of TGF beta sub(3) up-regulation of p27 super(Kip1) and p21 super(Cip1) by expression of RasN17 in epithelial cells

Our previous data demonstrated that Ras activation is necessary and sufficient for transforming growth factor beta (TGF beta )-mediated Erk1 activation, and is partially required for the inhibition of cyclin-dependent kinase 2 (Cdk2) activity, cyclin A expression and DNA synthesis by TGF beta . Here, we examined the kinetics and role of Ras in TGF beta sub(3)-mediated effects on specific G sub(1) cell cycle components in TGF beta -sensitive (4-1) and TGF beta -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 TGF beta sub(3) to up-regulate both CKI's. In contrast, the ability of TGF beta sub(3) to up-regulate p27 super(Kip1) and p21 super(Cip1) was maintained in ZnCl sub(2)-treated control cells. Inactivation of Ras also completely blocked the rapid TGF beta -mediated increase in new synthesis of p27 super(Kip1) protein. Moreover, up-regulation of p21 super(Cip1) protein levels and new synthesis of p27 super(Kip1), as well as the association of these CKI's with Cdk2, preceded the decrease in Cdk2 activity by TGF beta . Collectively, our results suggest that p21 super(Cip1) and p27 super(Kip1) are upstream effectors of the TGF beta -mediated inhibition of Cdk2 activity in IEC 4-1 cells, and demonstrate that Ras activation is obligatory for TGF beta -mediated up-regulation of these CKIs in untransformed epithelial cells.