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 设计新的治疗方法。我们发现了一种丝氨酸/苏氨酸 激酶，称为MAP激酶，其在胰岛素刺激的 蛋白激酶级联介导至少一些细胞作用， 胰岛素，包括其标志-糖原合成酶的激活。以限定 MAP激酶激活的上游途径，我们将重点放在 利用生物化学和分子遗传学 接近。 1. MAP激酶激酶（MAPKK）的cDNA的表征。我们有 分离并测序编码MAPKK的cDNA（K28）。此外该 分子克隆的策略允许分离额外的克隆， 其中至少一个（K5）编码另外的MAPKK相关蛋白。 将完成克隆的分析，所有不同的MAPKK同源物 其中， 2.利用MAPKK cDNA来产生用于机械调节所需的试剂。 问题研究需要开发几种试剂和程序 上游关键生物化学研究的实验室 途径：重组MAPKK、抗MAPKK抗体、定点 MAPKK的突变体。 3.胰岛素激活MAPKK的机制分析 胰岛素刺激的MAPKK磷酸化的完整细胞中的特征 细胞MAPKK蛋白质异质性基础的确定。 胰岛素刺激的激活剂的鉴定和纯化 MAPKK通过再活化测定。对MAPKK核目标的评估。 4. p21 ras在胰岛素激活MAPKK中作用的研究 一些研究指出p21 ras是MAP的上游成分， 激酶途径，并将追求：在体外系统的开发 在哺乳动物细胞中通过p21 ras激活MAP激酶和利用 鉴定和纯化p21 ras靶蛋白 负责激活MAPKK。 5.检查上游组件的可能关联/相互作用 MAP激酶通路与胰岛素受体底物1（IRS-1） （与Morris白色，Joslin糖尿病中心合作）酪氨酸 磷酸化的IRS-1被假设为信号的“对接”蛋白。 转导蛋白质，因此选择性检查关联 IRS-1与MAP激酶途径的组分的结合。

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 functions in an insulin-stimulated protein kinase cascade to mediate at least some of the cellular actions of insulin, including its hallmark-activation of glycogen synthase. To define the upstream pathways for activation of MAP kinase, we are focusing on the following questions, utilizing both biochemical and molecular genetic approaches. 1. Characterization of cDNAs for MAP Kinase Kinase (MAPKK). We have isolated and sequenced a cDNA (K28) that encodes a MAPKK. In addition, the strategy for molecular cloning allowed isolation of additional clones at least one of which (K5) encodes an additional MAPKK related protein. Analysis of the clones will be completed and all distinct MAPKK homologs therein characterized. 2. Utilization of MAPKK cDNAs to generate reagents needed for mechanistic studies. Development of several reagents and procedures are necessary preliminaries for critical biochemical investigations of the upstream pathways: recombinant MAPKK(s), anti-MAPKK antibodies, site-directed mutants of MAPKKs. 3. Analysis of Mechanism(s) of activation of MAPKK by insulin. Characterization of insulin-stimulated phosphorylation of MAPKK in intact cells. Determination of the basis of MAPKK protein heterogeneity. Identification and purification of insulin-stimulated activator(s) of MAPKK by reactivation assays. Assessment of nuclear targeting of MAPKK. 4. Investigation of the role of p21 ras in activation of MAPKK by insulin. Several studies point to p21 ras as an upstream component of the MAP kinase pathway and will be pursued by: development of an in vitro system for activation of MAP kinase by p21 ras in mammalian cells and utilization of the system to identify and purify the target protein for p21 ras responsible for activation of MAPKK. 5. Examination of possible association/interaction of upstream components of MAP kinase pathway with Insulin Receptor Substrate 1 (IRS-1) (collaborative with Morris White, Joslin Diabetes Center) Tyrosine phosphorylated IRS-1 is hypothesized to be a 'docking' protein for signal transducing proteins, and therefore selective examination of associations of IRS-1 with components of the MAP kinase pathway will be made.