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 以及设计新的治疗方法。我们鉴定出一种丝氨酸/苏氨酸 一种名为MAP的激酶，在胰岛素刺激下发挥作用 蛋白激酶级联信号转导至少部分细胞活动 胰岛素，包括它的标志--激活糖原合成酶。要定义 MAP激酶的上游激活途径，我们关注的是 以下问题，利用生化和分子遗传学 接近了。 1.MAP激酶(MAPKK)的cDNA特征我们有 分离并测序了一个编码MAPKK的cDNA(K28)。此外， 分子克隆策略允许在以下位置分离其他克隆 其中至少一个(K5)编码额外的MAPKK相关蛋白。 将完成对克隆的分析和所有不同的MAPKK同源物 其中刻画了。 2.利用MAPKK cDNA产生机械所需的试剂 学习。开发几种试剂和程序是必要的 上游关键生化调查的初步研究 途径：重组MAPKK(S)，抗MAPKK抗体，定点 MAPKK的突变体。 3.胰岛素激活MAPKK的机制分析(S)。 胰岛素刺激的完整人MAPKK的磷酸化特征 细胞。MAPKK蛋白异质性基础的测定。 胰岛素刺激激活剂S的鉴定与纯化 MAPKK再激活实验。对MAPKK核靶向的评估。 4.探讨p21ras在胰岛素激活MAPKK中的作用。 一些研究指出，p21ras是MAP的上游组成部分。 一种体外系统的研究进展 P21ras在哺乳动物细胞中激活MAP激酶及其应用 P21ras靶蛋白的鉴定和纯化系统的建立 负责MAPKK的激活。 5.检查上游组件的可能关联/相互作用 胰岛素受体底物1(IRS-1)对MAP激酶通路的影响 (与乔斯林糖尿病中心莫里斯·怀特合作)酪氨酸 磷酸化的IRS-1被认为是一种信号对接蛋白 转导蛋白质，因此选择性地检查关联性 将对IRS-1与MAP激酶途径的组件进行比较。

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.