Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition.
In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor.
Kahan
C
C
Centre de Biochimie, Centre National de la Recherche Scientifique, Faculté des Sciences, Nice, France.
Seuwen
K
K
Meloche
S
S
Pouysségur
J
J
D000067956:Adenylyl Cyclase Inhibitors; D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D037342:Pertussis Toxin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D013917:Thrombin / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D010716:Phosphatidylinositols; D010566:Virulence Factors, Bordetella; D016222:Fibroblast Growth Factor 2; D015240:Phorbol 12,13-Dibutyrate; D002217:Carbachol; D037342:Pertussis Toxin; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013917:Thrombin; D000527:Alprostadil