REGULATION AND FUNCTION OF INSULIN STIMULATED MAP KINASE

对胰岛素的生化途径有全面的了解 信号转导是理解糖尿病发病机制的关键 以及设计新的疗法。我们鉴定出一种丝氨酸/苏氨酸激酶， 被称为MAP激酶，在3T3-S6激酶之前被胰岛素激活- L1细胞。我们将广泛使用我们为RAPID开发的程序 活化的MAPK的分离和稳定 蛋白水解酶和磷酸酶。我们重点解决以下几个问题： 1.胰岛素调节MAP激酶活性的机制分析 相当大的努力将被用来分析磷酸化在细胞内的作用 这项规定。(A)磷酸酶使活化的MAP激酶失活 将在体外进行研究。(B)MAP激酶失活(分离自 32PO4标记的细胞)的变化将与磷酸酶的变化相关 磷酸氨基酸含量。(C)重新激活地图的不同战略 酪氨酸特异性激酶(以前被磷酸酶灭活) 和/或丝氨酸/酪氨酸氨基转移酶将被研究。(D)地图的关联性 激酶与丝裂原刺激酪氨酸的40 kDa底物 将进行磷酸化测试。 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， 丝裂原激活的级联反应中MAP激酶的可能参与 S6激酶的激活。(A)丝氨酸使S6K II失活- MAP激酶的特异性磷酸酶将在体外进行研究。(B) 将用胰酶研究S6 KII中MAP激酶的磷酸化位点 多肽图谱。(C)卵母细胞中MAP激酶的激活 胰岛素将作为MAP激酶参与的前提条件进行研究 非洲爪哇卵母细胞在胰岛素刺激下S6KII的激活。(D)活体内 MAP激酶对S6 KII的磷酸化将通过比较 从卵母细胞中分离的S6 KII胰酶多肽图谱 胰岛素对S6KII的体外磷酸化与MAP激酶结合。 3.MAP激酶的进一步鉴定和纯化。(A)企图 将提供额外的证据，证明40kD的磷蛋白是 通过复性从十二烷基硫酸钠凝胶中洗脱出来的酶，叠氮三磷酸腺苷 MAPK活性的标记、免疫沉淀和标记 Ppp40+抗Py-Ig G。(B)将寻找MAP激酶的前体。 (C)将开发方法从 用于生产抗肽抗体的微测序的大宗来源 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.