De Fea K, Roth R A
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
Biochemistry. 1997 Oct 21;36(42):12939-47. doi: 10.1021/bi971157f.
Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.
本研究发现，人肾成纤维细胞（293）中内源性蛋白激酶C的激活会抑制胰岛素随后刺激所表达的胰岛素受体底物-1酪氨酸磷酸化的能力。如果胰岛素受体及其底物均从蛋白激酶C已被激活的细胞中分离出来，那么在体外磷酸化反应中也会观察到这种抑制作用。为了测试胰岛素受体底物-1的丝氨酸磷酸化是否参与了这一过程，将该分子的丝氨酸612突变为丙氨酸。结果发现，所表达的突变体的胰岛素刺激的酪氨酸磷酸化及相关的磷脂酰肌醇3激酶活性与所表达的野生型底物相当。然而，与野生型蛋白不同，蛋白激酶C的激活并未抑制S612A突变体的胰岛素刺激的酪氨酸磷酸化，也未抑制其随后与磷脂酰肌醇3激酶的结合。对体内标记的IRS-1和S612A突变体进行胰蛋白酶肽图谱分析显示，佛波酯（PMA）刺激野生型IRS-1中一个肽段的磷酸化，而该肽段在S612A突变体的胰蛋白酶肽图谱中不存在。此外，发现含有该磷酸丝氨酸及其附近酪氨酸的合成肽被胰岛素受体磷酸化的程度远低于不含磷酸丝氨酸的相同肽段。发现蛋白激酶C的激活使一种胞质激酶磷酸化该合成肽以及完整的胰岛素受体底物-1的能力提高了10倍。最后，与瘦小鼠同窝仔鼠肝脏提取物相比，ob/ob小鼠肝脏的胞质提取物显示，能够磷酸化该合成肽的激酶活性增加了8倍。这些结果表明，蛋白激酶C的激活刺激了一种激酶，该激酶可在丝氨酸612处磷酸化胰岛素受体底物-1，从而导致细胞内胰岛素信号传导受到抑制，这在某些肥胖模型中构成了胰岛素抵抗的潜在机制。
