Tyrosine Dephosphorylation and Deactivation of Insulin Receptor Substrate-1 by Protein-tyrosine Phosphatase 1B

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 g per min), and the enzyme activities varied over a range of 5.5 ؋ 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. Cellular protein-tyrosine phosphatases (PTPases)1 play a crucial role in maintaining the steady-state phosphotyrosine content of proteins in the insulin action pathway (1, 2). Much of the available evidence characterizing the role of PTPases in insulin signaling has focused on the regulation of the activation state of the insulin receptor itself, where several PTPases, in particular the intracellular enzyme PTP1B and the transmembrane homologs LAR and LRP (RPTP-␣), have been postulated to have a regulatory function (3-6). However, the identity of PTPase(s) that regulate the tyrosine phosphorylation state of substrates of the insulin receptor kinase, such as IRS-1, has not been determined. IRS-1 is phosphorylated on multiple tyrosine residues by the activated insulin receptor kinase and serves as a docking site or scaffold for a series of adaptor proteins that possess Src homology 2 (SH2) domains (7). These proteins bind to specific phosphotyrosine sites on IRS-1, become activated, and transmit the downstream signal from the insulin receptor to various met...

Protein-tyrosine-phosphatase 1B activation is regulated developmentally in muscle of neonatal pigs

“…PTP1B activity in muscle, but not liver, was lower in 7-compared with 26-day-old pigs (* P Ͻ 0.05). Because recent studies (21,35) indicate that the binding of Grb2 adaptor protein with PTP1B is important in forming the PTP1B-Grb2-IRS-1 ternary complex, followed by dephosphorylation of IRS-1, we evaluated the effect of development on the association of PTP1B with the Grb2 adaptor protein. Equal amounts of skeletal muscle and liver extracts were immunoprecipitated with anti-Grb2 antibody, followed by immunoblotting with an anti-PTP1B antibody.…”

“…Finally, PTP1B undergoes auto-dephosphorylation and returns to its inactive basal state (12). There is some evidence that the steady-state phosphotyrosine content of IRS-1 is also regulated by PTP1B (21,35). In vitro, PTP1B, IRS-1, and Grb2 form a ternary complex, resulting in the dephosphorylation of IRS-1.…”

“…In vitro, PTP1B, IRS-1, and Grb2 form a ternary complex, resulting in the dephosphorylation of IRS-1. Therefore, the interaction between Grb2 and PTP1B, with subsequent effects on IRS-1 dephosphorylation, may also have an important role in the regulation of the postreceptor insulin-signaling pathway (21).…”

Increased Protein Tyrosine Phosphatase 1B (PTP1B) Activity and Cardiac Insulin Resistance Precede Mitochondrial and Contractile Dysfunction in Pressure‐Overloaded Hearts

“…Given that PTP1B may also dephosphorylate IRS1,38 we speculate that IRS1 phosphorylation may be affected by PTP1B leading to cIR at 10 weeks. In this study, we were not able to verify this possibility because of the complex regulation of IRS1, which includes at least 10 phosphorylation sites 20…”

A sesquiterpene quinone, dysidine, from the sponge Dysidea villosa, activates the insulin pathway through inhibition of PTPases

“…Protein tyrosine phosphatase 1B (PTP1B) was demonstrated to negatively regulate the insulin pathway via inactivation of IR and IRS1 [14][15][16] . Recently, the leptin pathway was also found to be regulated by PTP1B, in which the neuronal PTP1B binds and dephosphorylates JAK2, which was downstream of the leptin receptor, and subsequently inhibited leptin signaling [17,18] .…”