Goldstein B J, Bittner-Kowalczyk A, White M F, Harbeck M
Dorrance H. Hamilton Research Laboratories, Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 2000 Feb 11;275(6):4283-9. doi: 10.1074/jbc.275.6.4283.
Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular interactions that influence the balance of these processes, especially the phosphorylation state of postinsulin receptor substrates, such as insulin receptor substrate-1 (IRS-1). The specific activity of four candidate protein-tyrosine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-containing PTPase-2 (SHP-2), leukocyte common antigen-related (LAR), and leukocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro. PTP1B exhibited the highest specific activity (percentage dephosphorylated per microg per min), and the enzyme activities varied over a range of 5.5 x 10(3). When evaluated as a ratio of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B remained significantly more active by 3.1-293-fold, respectively. Overlay blots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showed that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit of phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 dephosphorylation. Further studies revealed that the adaptor protein GRB2 strongly promoted the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immunoprecipitation from an equimolar solution by 13.5 +/- 3.3-fold (n = 7; p < 0.01). Inclusion of GRB2 in a reaction mixture of IRS-1 and active PTP1B also increased the overall rate of IRS-1 tyrosine dephosphorylation by 2.7-3.9-fold (p < 0.01). These results provide new insight into novel molecular interactions involving PTP1B and GRB2 that may influence the steady-state capacity of IRS-1 to function as a phosphotyrosine scaffold and possibly affect the balance of postreceptor insulin signaling.
胰岛素受体及其受体后底物的稳态酪氨酸磷酸化调节是胰岛素信号转导的重要决定因素。然而，对于影响这些过程平衡的分子相互作用，尤其是胰岛素受体后底物（如胰岛素受体底物-1，IRS-1）的磷酸化状态，我们了解甚少。利用重组蛋白在体外研究了四种候选蛋白酪氨酸磷酸酶（蛋白酪氨酸磷酸酶1B（PTP1B）、含SH2结构域的蛋白酪氨酸磷酸酶-2（SHP-2）、白细胞共同抗原相关（LAR）和白细胞抗原相关磷酸酶（LRP））对IRS-1去磷酸化的比活性。PTP1B表现出最高的比活性（每分钟每微克去磷酸化的百分比），酶活性在5.5×10³范围内变化。当以与IRS-1的活性与与对硝基苯磷酸的活性之比进行评估时，PTP1B分别显著更具活性3.1至293倍。用IRS-1衔接蛋白的重组Src同源2结构域进行的印迹覆盖显示，Crk、GRB2、SHP-2和磷脂酰肌醇3'-激酶的p85亚基与IRS-1结合的丧失与IRS-1总体去磷酸化速率平行。进一步的研究表明，衔接蛋白GRB2强烈促进酪氨酸磷酸化的IRS-1与催化无活性的PTP1B之间形成稳定的蛋白复合物，使它们从等摩尔溶液中的共免疫沉淀增加了13.5±3.3倍（n = 7；p < 0.01）。在IRS-1和活性PTP1B的反应混合物中加入GRB2也使IRS-1酪氨酸去磷酸化的总体速率提高了2.7至3.9倍（p < 0.01）。这些结果为涉及PTP1B和GRB2的新型分子相互作用提供了新的见解，这些相互作用可能影响IRS-1作为磷酸酪氨酸支架的稳态能力，并可能影响受体后胰岛素信号的平衡。
