Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity.

We have examined the phosphorylation and protein kinase activity of p44 mitogen-activated protein kinase (p44mapk) in growth factor-stimulated hamster fibroblasts using a specific antiserum. The activity of p44mapk was stimulated both by receptor tyrosine kinases and G protein-coupled receptors. Detailed kinetics revealed that alpha-thrombin induces a biphasic activation of p44mapk in CCL39 cells: a rapid phase appearing at 5-10 min was followed by a late and sustained phase still elevated after 4 h. Inactivation of alpha-thrombin with hirudin after 30 sec, which prevented DNA synthesis, did not alter the early p44mapk response but completely abolished the late phase. Pretreatment of the cells with pertussis toxin, which inhibits by more than 95% alpha-thrombin-induced mitogenicity, resulted in the complete loss of late phase activity, while the early peak was partially attenuated. Treatment of CCL39 cells with basic fibroblast growth factor also induced a strong activation of p44mapk. Serotonin, which is not a mitogen by its own, had no effect on late phase p44mapk activity, but synergized with basic fibroblast growth factor to induce late kinase response and DNA synthesis. Both early and late phase activation of p44mapk were accompanied by tyrosine phosphorylation of the enzyme. Together, the results indicate that there is a very close correlation between the ability of a growth factor to induce late and sustained p44mapk activation and its mitogenic potential. Therefore, we propose that sustained p44mapk activation is an obligatory event for growth factor-induced cell cycle progression.

[1]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[2]  T. Hunter,et al.  Detection and quantification of phosphotyrosine in proteins. , 1983, Methods in enzymology.

[3]  J. Pouysségur,et al.  alpha‐Thrombin‐induced early mitogenic signalling events and G0 to S‐phase transition of fibroblasts require continual external stimulation. , 1985, The EMBO journal.

[4]  J. Pouysségur,et al.  Pertussis toxin inhibits thrombin‐induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts. , 1986, The EMBO journal.

[5]  J. Hofsteenge,et al.  Kinetics of the inhibition of thrombin by hirudin. , 1986, Biochemistry.

[6]  J. Pouysségur,et al.  Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin , 1987, Nature.

[7]  J. Maller,et al.  Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II , 1988, Nature.

[8]  E. Krebs,et al.  Activation of multiple protein kinases during the burst in protein phosphorylation that precedes the first meiotic cell division in Xenopus oocytes. , 1988, The Journal of biological chemistry.

[9]  J. Pouysségur,et al.  Serotonin stimulates DNA synthesis in fibroblasts acting through 5–HT1B receptors coupled to a Gi-protein , 1988, Nature.

[10]  J. Pouysségur,et al.  Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C. , 1988, The Biochemical journal.

[11]  D. Fabbro,et al.  EGF induces biphasic S6 kinase activation: Late phase is protein kinase C-dependent and contributes to mitogenicity , 1989, Cell.

[12]  J. Thorner,et al.  A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae , 1989, Cell.

[13]  J. Pouysségur,et al.  Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors. , 1990, The Journal of biological chemistry.

[14]  G. Fink,et al.  FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation , 1990, Cell.

[15]  C. Slaughter,et al.  An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. , 1990, Science.

[16]  J. Maller,et al.  Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase , 1990, Nature.

[17]  M. Caron,et al.  Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene. , 1990, Cell regulation.

[18]  E. Krebs,et al.  Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. , 1990, The Journal of biological chemistry.

[19]  A E Nel,et al.  Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C. , 1990, Journal of immunology.

[20]  Nancy Y. Ip,et al.  ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF , 1991, Cell.

[21]  T. Wright,et al.  Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation. , 1991, Cell regulation.

[22]  M. Cobb,et al.  Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. , 1991, Cell regulation.

[23]  J. Shabanowitz,et al.  Identification of the regulatory phosphorylation sites in pp42/mitogen‐activated protein kinase (MAP kinase). , 1991, The EMBO journal.

[24]  M. Villereal,et al.  Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation. , 1991, Cell regulation.

[25]  R. Erikson,et al.  Structure, expression, and regulation of protein kinases involved in the phosphorylation of ribosomal protein S6. , 1991, The Journal of biological chemistry.

[26]  Jie Wu,et al.  Sequence of pp42/MAP kinase, a serine/threonine kinase regulated by tyrosine phosphorylation , 1991, Nucleic Acids Res..

[27]  S. Pelech,et al.  Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. , 1991, The Journal of biological chemistry.

[28]  E. Krebs,et al.  Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.