A complete understanding of the biochemical pathways used for insulin signalling is essential for understanding pathogenesis of diabetes mellitus and designing new therapies. We identified a serine/threonine kinase, termed MAP kinase, which is activated by insulin prior to S6 kinase in 3T3- L1 cells. Extensive use will be made of a procedure we developed for rapid isolation and stabilization of activated MAP kinase, resolved from other kinases and phosphatases. We are focusing on the following questions: 1. Analysis of Mechanisms of Regulation of MAP Kinase Activity by Insulin. Considerable effort will go into analyzing the role of phosphorylation in this regulation. (a) Inactivation of activated MAP kinase by phosphatases will be studied in vitro. (b) Inactivation of MAP kinase (isolated from 32PO4 -labelled cells) by phosphatase will be correlated with changes in phosphoamino acid content. (c) Different strategies for reactivating MAP kinase (previously inactivated by phosphatase) by tyrosine specific kinases and/or Ser/thr kinases will be investigated. (d) The relatedness of MAP kinase and the 40 kDa substrate for mitogen stimulated tyrosine phosphorylation will be tested. 2. Analysis of Cellular Functions of MAP Kinase. In this regard, most of our attention will be focused on expanding our collaborative investigation, of the possible involvement of MAP kinase in a mitogenic cascade for activation of S6 kinase. (a) Activation of S6K II inactivated by serine- specific phosphatases by MAP kinase will be studied in vitro. (b) The phosphorylation sites in S6 KII for MAP kinase will be studied by tryptic peptide mapping. (c) Activation of MAP kinase in oocytes stimulated with insulin will be studied as a precondition for involvement of MAP kinase in S6 KII activation in Xenopus oocytes stimulated with insulin. (d) In vivo phosphorylation of S6 KII by MAP kinase will be tested by comparison of tryptic peptide maps of S6 KII isolated from oocytes stimulated with insulin to S6 KII phosphorylated in vitro with MAP kinase. 3. Further Characterization and Purification of MAP Kinase. (a) Attempts will be made to provide additional proof that the 40 kD a phosphoprotein is the kinase by renaturation of the band eluted form SDS gels, azido-ATP labelling, and immunoprecipitation of MAP kinase activity and labelled ppp40 with anti-pY IgG. (b) A precursor for MAP kinase will be sought. (c) Methods will be developed for isolation of sufficient MAP kinase from a bulk source for microsequencing for production of anti-peptide antibodies and nucleic acid probes.
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