Protein tyrosine phosphatases

Insulin receptor signal transduction plays a critical role in regulating pancreatic β-cell function, notably the acute first-phase insulin release in response to glucose. The basis for insulin resistance in pancreatic β-cells is not well understood but may be related to abnormal regulation of tyrosine phosphorylation events, which, in turn, may alter organization of insulin-signaling molecules in space and time. Members of the protein tyrosine phosphatase (PTPase) family are both functionally and structurally diverse; and within the past few years data have emerged from many laboratories that suggest selectivity of the PTPase catalytic domains toward cellular substrates. Of significance, a subset of PTPases has been implicated in the regulation of insulin signaling in a number of insulin-sensitive tissues. Alteration in PTPase expression or activity has been associated with abnormal regulation of tyrosine phosphorylation events and is accompanied by modulation of insulin sensitivity in vivo. Manipulations aimed at reducing expression of physiologically relevant PTPases acting at a step proximal to the insulin receptor are accompanied by normalization of blood glucose levels and improved insulin sensitivity in both normal and diabetic animals. Hence, the development of tissue-specific gene inactivation strategies should facilitate the study of the potential role of PTPases in β-cell insulin signaling transduction.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.     

Protein-tyrosine phosphatases and the regulation of insulin action.

Protein-tyrosine phosphatases (PTPases) play an important role in the regulation of insulin action by dephosphorylating the active (autophosphorylated) form of the insulin receptor and attenuating its tyrosine kinase activity. PTPases can also modulate post-receptor signalling by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase. Dramatic advances have recently been made in our understanding of PTPases as an extensive family of transmembrane and intracellular proteins that are involved in a number of pathways of cellular signal transduction. Identification of the PTPase(s) which act on various components of the insulin action cascade will not only enhance our understanding of insulin signalling but will also clarify the potential involvement of PTPases in the pathophysiology of insulin-resistant disease states. This brief review provides a summary of reversible tyrosine phosphorylation events in insulin action and available data on candidate PTPases in liver and skeletal muscle that may be involved in the regulation of insulin action.     

Cell density-dependent changes in the insulin action pathway: Evidence for involvement of protein-tyrosine phosphatases

In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high M_r cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 10⁵ to 3.2 × 10⁶ per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high M_r receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high M_r proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain.

A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity. We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B. Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system. When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03). To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases. Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01). The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.     

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.     

Protein Tyrosine Phosphatase 1B is Impaired in Skeletal Muscle of Diabetic Psammomys Obesus

Protein tyrosine phosphatases (PTPases) have been suggested to modulate the insulin receptor signal transduction pathways.We studied PTPases in Psammomys obesus, an animal model of nutritionally induced insulin resistance. No changes in the protein expression level of src homology PTPase 2 (SHP-2) (muscle, liver) or leukocyte antigen receptor (LAR) (liver) were detected. In contrast, the expression level of PTPase 1B (PTP 1B) in the skeletal muscle, but not in liver, was increased by 83% in the diabetic animals, compared with a diabetes-resistant line. However, PTP 1B– specific activity (activity/protein) significantly decreased (50% to 56%) in skeletal muscle of diabetic animals, compared with both the diabetes-resistant line and diabetes-prone animals. In addition, PTP 1B activity was inversely correlated to serum glucose level (r = –.434, P < .02). These findings suggest that PTP 1B, though overexpressed, is not involved in the susceptibility to insulin resistance in Psammomys obesus and is secondarily attenuated by hyperglycemia or other factors in the diabetic milieu.     

Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction

Insulin signaling involves a dynamic cascade of protein tyrosine phosphorylation and dephosphorylation. Most of our understanding of this process comes from studies focusing on tyrosine kinases, which are signal activators. Our knowledge of the role of protein-tyrosine phosphatases (PTPases), signal attenuators, in regulating insulin signal transduction remains rather limited. Protein-tyrosine phosphatase 1B (PTP-1B), the prototypical PTPase, is ubiquitously and abundantly expressed. Work from several laboratories, including our own, has implicated PTP-1B as a negative regulator of insulin action and as a potentially important mediator in the pathogenesis of insulin-resistance and non-insulin dependent diabetes mellitus (NIDDM).     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases

Protein-tyrosine phosphatases (PTPases) have been implicated in the physiological regulation of the insulin signalling pathway. In cellular and molecular studies, the transmembrane, receptor-type PTPase LAR and the intracellular, non-receptor enzyme PTP1B have been shown to have a direct impact on insulin action in intact cell models. Since insulin signalling can be enhanced by reducing the abundance or activity of specific PTPases, pharmaceutical agents directed at blocking the interaction between individual PTPases and the insulin receptor may have potential clinical relevance to the treatment of insulin-resistant states such as obesity and Type II diabetes mellitus.     

Transient Association of the Phosphotyrosine Phosphatase SHP-2 with TrkA Is Induced by Nerve Growth Factor

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP-2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP-2 with TrkA was accompanied by the tyrosine phosphorylation of SHP-2 and an association of SHP-2 with multiple tyrosine-phosphorylated proteins. In addition, the PTPase activity in SHP-2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP-2 with TrkA is induced by NGF and that this association leads to SHP-2 activation and tyrosine phosphorylation. We conclude that SHP-2 plays a significant role in early biochemical events in TrkA-mediated signal transduction.     

Structural diversity of eukaryotic protein tyrosine phosphatases: functional and evolutionary implications

In the past few years, very rapid advances have been made in determining the primary structure of protein tyrosine phosphatases (PTPases). PTPase genes have now been isolated from bacteria, viruses, yeasts and insects as well as vertebrates. The cytosolic PTPases have a catalytic domain associated with various accessory domains that are believed to be involved in protein-protein interaction or subcellular localization. The transmembrane PTPases have either one or two cytoplasmic PTPase domains and an extracellular receptor-like structure. The existence of a large number of structurally diverse PTPases suggests that they play specific and crucial roles in signal transduction. In this article, the structural features of the PTPases from higher eukaryotes are reviewed.     

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.     

Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.     

Phosphotyrosyl turnover in insulin signaling. Characterization of two membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1 adipocytes.

It was shown previously that 422 (aP2) protein, a 15-kDa fatty acid binding protein, is phosphorylated on Tyr19 both in vitro by the insulin receptor tyrosine kinase and in intact 3T3-L1 adipocytes treated with insulin and phenylarsine oxide (PAO). Phospho-422(aP2) protein (pp15) accumulates in cells treated with insulin and PAO because the arsenical blocks turnover of the phosphoryl group of pp15. These findings suggest that a PAO-sensitive enzyme mediates turnover of the pp15 tyrosine phosphoryl group. We have purified and characterized two membrane protein tyrosine phosphatases (PTPases) from 3T3-L1 adipocytes that catalyze hydrolysis of phospho-Tyr19 of authentic pp15. These enzymes, designated PTPases HA1 and HA2, were purified approximately 20,000-fold and approximately 15,000-fold, respectively, and shown to differ markedly in their sensitivity to both vanadate and phosphotyrosine. Both enzymes are inhibited by PAO and accordingly can be labeled with 4-[125I]iodo-PAO. By this method, it was demonstrated that PTPases HA1 and HA2 have molecular masses of approximately 60 kDa and approximately 38 kDa, respectively. Both enzymes exhibit substrate preference for pp15 when compared with other phosphotyrosine-containing protein substrates. Proteins containing phosphoserine and phosphothreonine do not serve as substrates for the enzymes. The pp15 PTPase HA2 is expressed both in 3T3-L1 preadipocytes and adipocytes, whereas pp15 PTPase HA1 is expressed only in 3T3-L1 adipocytes.     

cDNA cloning of rat LRP, a receptor like protein tyrosine phosphatase, and evidence for its gene regulation in cultured rat mesangial cells.

Protein tyrosine phosphatases (PTPases) are a family of enzymes that play a crucial role in the regulation of signal transduction mediated by reversible protein tyrosine phosphorylation. To understand the significance of PTPases in physiological and pathophysiological processes in the kidney, we isolated three cDNA segments encoding PTPases (LAR, LRP and a novel PTPase) from rat kidney by polymerase chain reaction (PCR). Using PCR product as a probe, we isolated a full-length cDNA of rat LRP. LRP cDNA encoded a single membrane spanning protein consisted of 796 amino acids, with two tandemly located intracellular PTPase domains. By Northern analysis, a ubiquitous pattern of LRP gene expression in rat tissues was demonstrated. In cultured rat mesangial cells, LRP mRNA was detected and the mRNA level was suppressed by either interleukin-1 or interleukin-6 treatment.