Kelly K L, Ruderman N B
Boston University Medical Center, Evans Department of Medicine, Massachusetts 02118-2393.
J Biol Chem. 1993 Feb 25;268(6):4391-8.
Insulin stimulates the appearance of anti-tyrosine(P)-immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity in adipocytes, predominantly in an intracellular membrane fraction (Kelly, K. L., Ruderman, N. B., and Chen, K. S. (1992) J. Biol. Chem. 267, 3423-3428). Neither the mechanism underlying this activation nor the precise subcellular compartment in which it occurs is known. To address these questions, studies were performed using isolated rat adipocytes and subcellular fractions of these cells. In intact cells, insulin stimulated the rapid appearance of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in 32P-labeled adipocytes without changing the labeling of phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate. This effect was accompanied by the tyrosyl phosphorylation of a 185-kDa protein, tentatively identified as IRS-1, with which PI 3-kinase became associated. The majority of the p85, the regulatory subunit of PI 3-kinase, in untreated adipocytes was present in the cytosol; however, neither the activity of PI 3-kinase nor the total amount of p85 in this fraction was modified in response to insulin. In contrast, insulin increased the association of p85 with IRS-1, the tyrosyl phosphorylation of the IRS-1 associated with p85, and the total activity of PI 3-kinase in the plasma membranes and low density membranes. After insulin treatment, similar amounts of p85 were bound to IRS-1 in the low density and plasma membrane fractions; however, tyrosyl-phosphorylated IRS-1 and PI 3-kinase activity were an order of magnitude greater in the low density membranes. The complex of tyrosyl-phosphorylated IRS-1.p85 that formed in response to insulin was localized to a very low density vesicle subpopulation that could be distinguished from vesicles containing the GLUT-4 glucose transporter and the insulin receptor. These data suggest that the activation of PI 3-kinase by insulin in the adipocyte involves the formation of a complex between IRS-1 and PI 3-kinase in a very low density membrane fraction that is not enriched in GLUT-4 or insulin receptors. They also suggest that PI 3-kinase activation correlates more closely with the extent of tyrosyl phosphorylation of the IRS-1 complexed to PI 3-kinase than it does to the amount of p85 bound to IRS-1.
胰岛素可刺激脂肪细胞中抗酪氨酸（磷酸化）免疫沉淀的磷脂酰肌醇3激酶（PI 3激酶）活性的出现，主要出现在细胞内膜部分（凯利，K.L.，鲁德曼，N.B.，和陈，K.S.（1992年）《生物化学杂志》267卷，3423 - 3428页）。这种激活作用的潜在机制及其发生的确切亚细胞区室均尚不清楚。为了解决这些问题，我们使用分离的大鼠脂肪细胞及其亚细胞组分进行了研究。在完整细胞中，胰岛素刺激32P标记的脂肪细胞中磷脂酰肌醇3,4 - 二磷酸和磷脂酰肌醇3,4,5 - 三磷酸迅速出现，而不改变磷脂酰肌醇3 - 磷酸、磷脂酰肌醇4 - 磷酸或磷脂酰肌醇4,5 - 二磷酸的标记情况。这种效应伴随着一种185 kDa蛋白的酪氨酸磷酸化，该蛋白初步鉴定为IRS - 1，PI 3激酶与之结合。未处理的脂肪细胞中，PI 3激酶的调节亚基p85大部分存在于胞质溶胶中；然而，该组分中PI 3激酶的活性和p85的总量均未因胰岛素而改变。相反，胰岛素增加了p85与IRS - 1的结合、与p85结合的IRS - 1的酪氨酸磷酸化以及质膜和低密度膜中PI 3激酶的总活性。胰岛素处理后，低密度膜和质膜组分中与IRS - 1结合的p85量相似；然而，低密度膜中酪氨酸磷酸化的IRS - 1和PI 3激酶活性高一个数量级。胰岛素刺激形成的酪氨酸磷酸化IRS - 1.p85复合物定位于一个极低密度的小泡亚群，该亚群可与含有GLUT - 4葡萄糖转运蛋白和胰岛素受体的小泡区分开来。这些数据表明，脂肪细胞中胰岛素对PI 3激酶的激活涉及IRS - 1与PI 3激酶在一个极低密度膜组分中形成复合物，该组分不富含GLUT - 4或胰岛素受体。它们还表明，PI 3激酶的激活与与PI 3激酶复合的IRS - 1的酪氨酸磷酸化程度的相关性比与与IRS - 1结合的p85量的相关性更紧密。
