Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B - Possible facilitation by the formation of a ternary complex with the GRB2 adaptor protein

Regulation of the steady-state tyrosine phosphorylation of the insulin rece ptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular inter actions that influence the balance of these processes, especially the phosp horylation state of postinsulin receptor substrates, such as insulin recept or substrate-1 (IRS-1). The specific activity of four candidate protein-tyr osine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-con taining PT-Pase-2 (SHP-2), leukocyte common antigen-related (LAR), and leuk ocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro, PTP1B exhibited the highest s pecific activity (percentage dephosphorylated per mu g per min), and the en zyme activities varied over a range of 5.5 x 10(3). When evaluated as a rat io of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B r emained significantly more active by 3.1-293-fold, respectively. Overlay bl ots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showe d that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit o f phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 depho sphorylation, Further studies revealed that the adaptor protein GRB2 strong ly promoted the formation of a stable protein complex between tyrosine-phos phorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immu noprecipitation 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 a lso 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 c apacity of IRS-1 to function as a phosphotyrosine scaffold and possibly aff ect the balance of postreceptor insulin signaling.