Molecular mechanisms of insulin signal transduction and its disorder

在将胰岛素加入过表达胰岛素受体的细胞后，抗磷酸酪氨酸免疫沉淀物中磷脂酰肌醇（PI）3-激酶的活性迅速且大大增加。因此，这种酶可能是胰岛素受体酪氨酸激酶的底物，也可能是胰岛素信号转导的介质之一。然而，尚不清楚胰岛素受体的活化酪氨酸激酶是否直接磷酸化PI 3-激酶的酪氨酸残基，以及胰岛素是否刺激PI 3-激酶的比活性。我们曾报道在体外胰岛素受体作用下PI 3-激酶85-kDa亚基的酪氨酸残基发生磷酸化，为了研究PI 3-激酶的酪氨酸磷酸化及其活性在体内胰岛素作用下的变化，我们用PI 3-激酶85-kDa亚基的特异性抗体进行了研究。PI 3-kinase活性在PI 3-kinase p85亚单位抗体免疫沉淀物中明显增强， ...更多信息 通过胰岛素处理过表达胰岛素受体的细胞，其表达量增加约3倍。胰岛素治疗还刺激了体内PI 3-激酶α-型85-kDa亚基的酪氨酸、丝氨酸和苏氨酸磷酸化。磷酸酶处理的免疫沉淀废除PI 3-激酶活性的增加。PI 3-激酶本身的磷酸化、相关蛋白的酪氨酸磷酸化或磷酸化PI 3-激酶与相关蛋白的复合物的形成都可能增加PI 3-激酶的活性。用<32>反相高效液相色谱和薄层电泳分析胰岛素处理后完整细胞中磷酸化α-型p85的赖氨酰内肽酶消化获得的磷酸肽。PI 3-激酶的α-型p85在酪氨酸368、580和607处被体内胰岛素受体磷酸化。少

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro.To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was inc … More reased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylations of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.In the next work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [^<32>P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo. Less