Heffetz D, Rutter W J, Zick Y
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Biochem J. 1992 Dec 1;288 ( Pt 2)(Pt 2):631-5. doi: 10.1042/bj2880631.
H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate or H2O2 alone. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO.T cells) several proteins (e.g. pp180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mM-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 microM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO.YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.
过氧化氢（H2O2）和钒酸盐是已知的类胰岛素剂。它们共同诱导胰岛素的生物效应，其效力超过单独使用胰岛素、钒酸盐或H2O2时的效果。我们之前已经表明，将H2O2和钒酸盐添加到完整细胞中时，由于这些试剂对细胞内蛋白酪氨酸磷酸酶（PTPases）的抑制作用，会迅速刺激蛋白酪氨酸磷酸化。使用抗磷酸酪氨酸抗体进行蛋白质印迹分析，我们现在已经在转染了野生型胰岛素受体基因的中国仓鼠卵巢（CHO）细胞（CHO.T细胞）中鉴定出几种蛋白质（例如pp180、125、100、60和52），在用胰岛素和3 mM - H2O2处理细胞后，其磷酸酪氨酸含量迅速增加。当100 microM偏钒酸钠与H2O2一起添加时，这些蛋白质以及其他蛋白质的酪氨酸磷酸化进一步增强。在用胰岛素受体基因转染的CHO细胞中，酪氨酸1162和1163被苯丙氨酸取代（CHO.YF - 3细胞），胰岛素、胰岛素/H2O2和H2O2/钒酸盐对酪氨酸磷酸化的影响明显降低。同样，在存在胰岛素或类胰岛素剂的情况下培养的亲本CHO细胞中，这些蛋白质中的大多数未能经历增强的酪氨酸磷酸化。我们的研究结果表明，H2O2/钒酸盐对PTPase活性的抑制增强了胰岛素受体激酶酪氨酸1162和1163的自磷酸化，导致其以胰岛素非依赖方式激活。结果，该酶的潜在靶标的酪氨酸磷酸化发生。H2O2/钒酸盐在缺乏这些双酪氨酸残基的受体突变体中未能诱导这些蛋白质的磷酸化，支持了这一假设。
