Dynamics of Protein-tyrosine Phosphatases in Rat Adipocytes

Abstract

Protein-tyrosine phosphatases (PTPases) play a key role in maintaining the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate proteins such as insulin receptor substrate 1 (IRS-1). However, the PTPase(s) that inactivate IR and IRS-1 under physiological conditions remain unidentified. Here, we analyze the subcellular distribution in rat adipocytes of several PTPases thought to be involved in the counterregulation of insulin signaling. We found that the transmembrane enzymes, protein-tyrosine phosphatase (PTP)-␣ and leukocyte common antigen-related (LAR), were detected predominantly in the plasma membrane and to a lesser extent in the heavy microsomes, a distribution similar to that of insulin receptor. PTP-1B and IRS-1 were present in light microsomes and cytosol, whereas SHPTP2/Syp was exclusively cytosolic. Insulin induced a redistribution of PTP-␣ from the plasma membrane to the heavy microsomes in a parallel fashion with the receptor. The distribution of PTP-1B in the light microsomes from resting adipocytes was similar to that of IRS-1 as determined by sucrose velocity gradient fractionation. Analysis of the catalytic activity of partially purified rat adipocyte PTP-␣ and LAR and recombinant PTP-1B showed that all three PTPases dephosphorylate IR. When a mix of IR/IRS-1 was used as a substrate, PTP-1B was particularly effective in dephosphorylating IRS-1. Considering that IR and IRS-1 can be dephosphorylated in internal membrane compartments from rat adipocytes (Kublaoui, B., Lee, J., and Pilch, P.F. (1995) J. Biol. Chem. 270, 59 -65) and that PTP-␣ and PTP-1B are the respective PTPases in these fractions, we conclude that these PTPases are responsible for the counterregulation of insulin signaling there, whereas both LAR and PTP-␣ may act upon cell surface insulin receptors.The initial steps in insulin signaling require protein tyrosine phosphorylation. The interaction of insulin with its plasma membrane receptor elicits a rapid autophosphorylation on specific tyrosine residues in several domains of the receptor's cytoplasmic ␤-subunit followed by activation of the receptor's exogenous tyrosine kinase activity as reflected by enhanced substrate phosphorylation (reviewed in Refs. 1 and 2). The principle insulin receptor substrates (IRSs) 1 involved in metabolic regulation are IRS-1 and IRS-2, and there is abundant biochemical evidence to support this (reviewed in Ref.3). Genetic evidence from animals devoid of IRS-1 (4, 5) and IRS-2 (6) further suggests that these molecules are intimately involved in the signaling to metabolic pathways regulated by insulin. Two additional members of the IRS family, IRS-3 and IRS-4, have been described, but they are unlikely to be involved in insulin-dependent metabolic regulation because the former is not expressed in the appropriate tissues (7) and a knockout of the latter has no obvious metabolic phenotype (8). IRS-1 and IRS-2 contain numerous tyrosine residues that, when phosphorylated, act as docking sites for a certain set of Src homolo...

Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance

“…Thus, although previous studies in cell lines indicate that LAR can dephosphorylate IR (16)(17)(18), our data suggest that in vivo, LAR acts more distally, perhaps by directly dephosphorylating specific sites on IRS proteins. This finding is consistent with the demonstration that IRS-1 is a substrate for LAR in vitro (19,20). However, we cannot exclude the possibility that LAR may dephosphorylate specific tyrosyl residues on IR that are not detected when using an assay of total IR tyrosyl phosphorylation.…”

“…In vitro studies suggest that LAR preferentially dephosphorylates insulin receptor Tyr-1150 (18), one of three tyrosyl residues that are critical for receptor activity (2). IRS-1 also is a substrate of LAR in vitro (19,20).…”

Inhibition of Insulin Receptor Catalytic Activity by the Molecular Adapter Grb14

“…7A). In agreement with recent studies (38), PTP1B strikingly decreased IR tyrosine phosphorylation (Fig. 7A, lanes 2 and 3).…”

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

“…The available data indicate that there are a number of other PTPases, including PTP1-␣, LAR, and SHP2 (9,20). Therefore, the contribution of these phosphatases in the regulation of insulin sensitivity in the neonate cannot be ruled out.…”

“…Protein-tyrosine-phosphatase 1B (PTP1B), a nontransmembrane phosphotyrosine phosphatase, has been shown to attenuate insulin signaling by catalyzing the dephosphorylation of the IR and IRS-1 (9,17). Early studies suggest that PTP1B blocks insulininduced S6 peptide phosphorylation and inhibits insulin-induced maturation of Xenopus oocytes (10,41).…”