REGULATION AND FUNCTION OF INSULIN STIMULATED MAP KINASE

完整了解用于胰岛素的生化途径 信号传导对于理解糖尿病的发病机理是必不可少的 设计新的疗法。 我们鉴定了一种丝氨酸/苏氨酸激酶， 称为MAP激酶，其在3 T3中先于S6激酶被胰岛素激活。 L1细胞。 将广泛使用我们开发的一种程序， 活化MAP激酶的分离和稳定化，从其它 激酶和磷酸酶。 我们正集中研究以下问题： 1.胰岛素调节MAP激酶活性的机制分析。 相当大的努力将进入分析磷酸化的作用， 这个规定。 (a)磷酸酶对活化MAP激酶的失活作用 将在体外进行研究。 (b)MAP激酶的失活（分离自 32 PO 4标记的细胞）将与 磷酸氨基酸含量 (c)激活MAP的不同策略 酪氨酸特异性激酶的激酶（先前被磷酸酶灭活） 和/或Ser/Thr激酶。 (d)MAP的相关性 激酶和丝裂原刺激酪氨酸的40 kDa底物 将测试磷酸化。 2. MAP激酶的细胞功能分析 在这方面，大多数 我们的注意力将集中在扩大我们的合作调查上， MAP激酶可能参与促有丝分裂级联， 激活S6激酶。 (a)S6 K II的活化被丝氨酸- 将在体外研究MAP激酶的特异性磷酸酶。 (b)的 将通过胰蛋白酶消化法研究MAP激酶在S6 KII中的磷酸化位点。 肽图谱 (c)激活卵母细胞MAP激酶的研究 将研究胰岛素作为MAP激酶参与 胰岛素刺激的非洲爪蟾卵母细胞中S6 KII激活。 (d)体内 MAP激酶对S6 KII的磷酸化作用将通过比较 从刺激的卵母细胞中分离的S6 KII的胰蛋白酶肽图谱 在体外用MAP激酶将胰岛素磷酸化为S6 KII。 3. MAP激酶的进一步鉴定和纯化。 (a)尝试 将提供额外的证据，证明40 kD的磷蛋白是 通过复性从SDS凝胶洗脱的条带，叠氮基-ATP 标记和MAP激酶活性的免疫沉淀以及标记的 ppp 40与抗-pY IgG。 (b)将寻找MAP激酶的前体。 (c)将开发用于分离足够的MAP激酶的方法， 用于生产抗肽抗体的微测序的批量来源 和核酸探针。

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.