Jyotirmoy Kusari

Protein-tyrosine Phosphatase 1B Is a Negative Regulator of Insulin- and Insulin-like Growth Factor-I-stimulated Signaling

To understand the physiological role of protein-tyrosine phosphatase 1B (PTPase 1B) in insulin and insulin-like growth factor-I (IGF-I) signaling, we established clonal cell lines overexpressing wild type or inactive mutant (C215S) PTPase 1B in cells overexpressing insulin (Hirc) or IGF-I (CIGFR) receptors. PTPase 1B overexpression in transfected cells was verified by immunoblot analysis with a monoclonal PTPase 1B antibody. Subfractionation of parental cells demonstrated that greater than 90% of PTPase activity was localized in the Triton X-100-soluble particulate (P1) cell fraction. PTPase activity in the P1 fraction of cells overexpressing wild type PTPase 1B was 3-6-fold higher than parental cells but was unaltered in all fractions from C215S PTPase 1B-containing cells. The overexpression of wild type and C215S PTPase 1B had no effects on intrinsic receptor kinase activity, growth rate, or general cell morphology. The effects of PTPase 1B overexpression on cellular protein tyrosine phosphorylation were examined by anti-phosphotyrosine immunoblot analysis. No differences were apparent under basal conditions, but hormone-stimulated receptor autophosphorylation and/or insulin receptor substrate tyrosine phosphorylation were inhibited in cells overexpressing wild type PTPase 1B and increased in cells expressing mutant PTPase 1B, in comparison with parental cells. Metabolic signaling, assessed by ligand-stimulated [14C]glucose incorporation into glycogen, was also inhibited in cells overexpressing active PTPase 1B and enhanced in cells containing C215S PTPase 1B. These data strongly suggest that PTPase 1B acts as a negative regulator of insulin and IGF-I signaling.

Protein-Tyrosine Phosphatase 1B Complexes with the Insulin Receptor in Vivo and Is Tyrosine-phosphorylated in the Presence of Insulin

In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR β-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.

Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase 1B protein content are associated with insulin action and resistance.

Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent diabetes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was reduced 21% (P < 0.05) and 22% (P < 0.005) in obese nondiabetic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose disposal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of approximately 10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPase1B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased enzyme activity may have a role in the insulin resistance of obesity and NIDDM.

Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region.

Protein-tyrosine phosphatases (PTPs) play a major role in regulating insulin signaling. Among the PTPs that regulate this signaling pathway, PTP1B plays an especially prominent role. PTP1B inhibits insulin signaling and has previously been shown to bind to the activated insulin receptor (IR), but neither the mechanism nor the physiological importance of such binding have been established. Here, we show that a previously undefined region in the N-terminal, catalytic half of PTP1B contributes to IR binding. Point mutations within this region of PTP1B disrupt IR binding but do not affect the catalytic activity of this phosphatase. This binding-defective mutant of PTP1B does not efficiently dephosphorylate the IR in cells, nor does it effectively inhibit IR signaling. These results suggest that PTP1B targets the IR through a novel binding element and that binding is required for the physiological effects of PTP1B on IR signal transduction.

Marked impairment of protein tyrosine phosphatase 1B activity in adipose tissue of obese subjects with and without type 2 diabetes mellitus

Protein tyrosine phosphatases (PTPs) are required for the dephosphorylation of the insulin receptor (IR) and its initial cellular substrates, and it has recently been reported that PTP-1B may play a role in the pathogenesis of insulin resistance in obesity and type 2 diabetes mellitus (DM). We therefore determined the amount and activity of PTP-1B in abdominal adipose tissue obtained from lean nondiabetic subjects (lean control (LC)), obese nondiabetic subjects (obese control (OC)), and subjects with both type 2 DM (DM2) and obesity (obese diabetic (OD)). PTP-1B protein levels were 3-fold higher in OC than in LC (1444 +/- 195 U vs 500 +/- 146 U (mean +/- SEM), P < .015), while OD exhibited a 5.5-fold increase (2728 +/- 286 U, P < .01). PTP activity was assayed by measuring the dephosphorylating activity toward a phosphorus 32-labeled synthetic dodecapeptide. In contrast to the increased PTP-1B protein levels, PTP-1B activity per unit of PTP-1B protein was markedly reduced, by 71% and 88% in OC and OD, respectively. Non-PTP-1B tyrosine phosphatase activity was comparable in all three groups. Similar results were obtained when PTP-1B activity was measured against intact human IR. A significant correlation was found between body mass index (BMI) and PTP-1B level (r = 0.672, P < .02), whereas BMI and PTP-1B activity per unit of PTP-1B showed a strong inverse correlation (r = -0.801, P < .002). These data suggest that the insulin resistance of obesity and DM2 is characterized by the increased expression of a catalytically impaired PTP-1B in adipose tissue and that impaired PTP-1B activity may be pathogenic for insulin resistance in these conditions.

Regulation of Growth Factor-Induced Signaling by Protein-Tyrosine-Phosphatases

Abstract The binding of a growth factor to its specific receptor catalyzes a complex cascade of intracellular signaling events, characterized by changes in the phosphorylation state of many key proteins. Among these phosphorylation events, tyrosine phosphorylation plays a prominent role in the transmission of postreceptor signals. The state of tyrosine phosphorylation is regulated by the actions of protein-tyrosine kinases (PTKs) and protein-tyrosine-phosphatases (PTPs). Dysregulation of either event can lead to abnormal cellular responses. PTPs generally act to regulate negatively—that is, to turn off—any signals generated by PTKs. However, this is not always the case, as seen by the phosphatase SHP-2, which can either be a positive or negative regulator of signal transduction depending on the particular cellular context. In addition, a novel family of dual specificity phosphatases has been recently discovered. These enzymes are capable of dephosphorylating phosphotyrosine and phosphothreonine/phosphoserine residues, and seem to play a significant role in attenuating the action of MAP kinases. Several themes appear throughout PTP regulation of growth factor signaling, including positive or negative regulation, importance of cell/tissue type, identity of the receptor activated, and subcellular localization. Although only a handful of PTPs have been identified, the present work done in elucidating their function has revealed their significance in the maintenance of normal physiological responses to growth factors.

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38MAPK.

Insulin-Receptor cDNA Sequence in NIDDM Patient Homozygous for Insulin-Receptor Gene RFLP

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment–length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both α- and β-subunits. No difference was found in the predicted amino acid sequence of the subjects insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12–amino acid insert in the α-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patients transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself. Consequently, the RFLP for which this patient is homozygous is not associated with a coding-sequence abnormality in the insulin-receptor gene.