Perturbation of EGF-activated MEK1 and PKB signal pathways by TGF-beta1 correlates with perturbation of EGF-induced cyclin D1 and DNA synthesis by TGF-beta1 in C3H 10T1/2 cells

In mouse C3H 10T1/2 cells, we previously reported that TGF-beta1 first delays and later potentiates EGF-induced DNA synthesis corresponding to an inhibition of EGF-induced cyclin D1 expression at t = 13 h. We report here that in accord with DNA synthesis kinetics, TGF-beta1 initially suppresses EGF-induced cyclin D1 expression then later releases the inhibition. Furthermore, TGF-beta1 also first decreases and later potentiates the levels of EGF-activated MEK1/MAPK and PKB, indicating the existence of cross talk between TGF-beta 1- and EGF-activated signal transduction pathways. PD98059, the specific inhibitor of MEK1, significantly blocks EGF-induced DNA synthesis, whereas wortmannin, the PI3K inhibitor, exerts a modest inhibitory effect, which suggests that the activation of MEK1-MAPK pathway plays a major role in EGF-induced DNA synthesis and the activation of PI3K-PKB pathway plays a minor role. Upon examination of mechanisms underlying the cross talk, it was discovered that application of TGF-beta1 triggers a rapid association between Raf-1 and catalytic subunits of PKA, which are reported to be able to inactivate Raf-1 upon activation. Therefore, TGF-beta1 may activate PKA to inhibit the EGF-activated MEK1-MAPK pathway. The wortmannin-sensitive phosphorylation at the thr(389) site is necessary for activation of p70s6K, an important kinase involved in mitogen-stimulated protein synthesis. Although we found that EGF-stimulated p70s6K phosphorylates through a MAPK-dependent and a MAPK-independent (wortmannin-sensitive) pathway, TGF-beta1 failed to block EGF-triggered phosphorylation of p70s6K at thr(389) and thr(421)/ser(424) sites, implying that PKB inhibition by TGF-beta1 may result from inhibition of PDK1 activity instead of inhibition of PI3K activity. These data also suggest that TGF-beta1 may selectively perturb certain EGF-activated MAPK pools.