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.
Yan
S
S
Department of Biochemistry, Roswell Park Cancer Institute, Buffalo, New York 14260, USA.
Krebs
S
S
Leister
K J
KJ
Wenner
C E
CE
D000818:Animals; D002460:Cell Line; D019938:Cyclin D1 / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000502:physiology*; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D011518:Proto-Oncogene Proteins; D016212:Transforming Growth Factor beta; D019938:Cyclin D1; D004815:Epidermal Growth Factor; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases