Abstract
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.
Highlights
Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction
While the presence of GRB2 significantly increased the overall dephosphorylation of insulin receptor substrate-1 (IRS-1) in these studies, these techniques did not detect an change in the relative ability of PTP1B to dephosphorylate the phosphotyrosine residues on IRS-1 that mediate the binding of the specific Src homology 2 (SH2) adaptor proteins tested, including GRB2, SHP-2, p85, and Crk under the in vitro conditions employed
In order to provide initial data to help identify the protein-tyrosine phosphatases (PTPases)(s) that regulate the tyrosine phosphorylation state of IRS-1, in the present work we examined the activity of four candidate PTPases (PTP1B, SHP-2, LAR, and LRP) toward the dephosphorylation of IRS-1 in vitro
Summary
Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. The dephosphorylation of specific phosphotyrosine adaptor sites by individual PTPases was evaluated by studying their effect on the binding of purified recombinant SH2 domains of the docking proteins SHP-2, p85, Crk, and GRB2.
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