Cellular PTPase Activity Research Articles

Protein-tyrosine phosphatase activity in human adipocytes is strongly correlated with insulin-stimulated glucose uptake and is a target of insulin-induced oxidative inhibition

Protein-tyrosine phosphatases (PTPases), in particular PTP1B, have been shown to modulate insulin signal transduction in liver and skeletal muscle in animal models; however, their role in human adipose tissue remains unclear. The uptake of 14C-D-glucose in response to 10 or 100 nmol/L insulin was measured in isolated subcutaneous adipocytes from subjects with a mean age of 44 years (range, 26 to 58) and mean body mass index (BMI) of 35.6 (range, 29.7 to 45.5). The endogenous activity of total PTPases and specifically of PTP1B in immunoprecipitates was measured in cell lysates under an inert atmosphere with and without added reducing agents. Using nonlinear regression analysis, higher BMI was significantly correlated with lower adipocyte glucose uptake (r = 0.73, P = .01) and with increased endogenous total PTPase activity (r = 0.64, P = .04). Correlation with waist circumference gave similar results. The endogenous total PTPase activity also strongly correlated with insulin-stimulated glucose uptake (R = .89, P < .0001); however, the activity of PTP1B was unrelated to the level of glucose uptake. Consistent with the insulin-stimulated oxidative inhibition of thiol-dependent PTPases reported for 3T3-L1 adipocytes and hepatoma cells, treatment of human adipocytes with 100 nmol/L insulin for 5 minutes lowered endogenous PTPase activity to 37% of control (P < .001), which was increased 25% by subsequent treatment with dithiothreitol in vitro. Cellular treatment with diphenyleneiodonium (DPI), an NADPH oxidase inhibitor that blocks the cellular generation of H2O2 and reduces the insulin-induced reduction of cellular PTPase activity, also diminished insulin-stimulated glucose uptake by 82% (P = .001). These data suggest that total cellular PTPase activity, but not the activity of PTP1B, is higher in more obese subjects and is negatively associated with insulin-stimulated glucose transport. The insulin-stimulated oxidative inhibition of PTPases may also have an important permissive role in the transmission of the insulin signal to glucose transport in human adipocytes.

Depot-Specific Variation in Protein-Tyrosine Phosphatase Activities in Human Omental and Subcutaneous Adipose Tissue: A Potential Contribution to Differential Insulin Sensitivity

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42–71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor β-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

Depot-specific variation in protein-tyrosine phosphatase activities in human omental and subcutaneous adipose tissue: a potential contribution to differential insulin sensitivity.

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42-71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor beta-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

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.

Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells.

SPECIFIC AIMSProtein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is required for enzymatic activity. In this study, we evaluated how a strictly anaerobic working environment with deoxygenated buffers and avoiding exposure to air permits the assessment of endogenous PTPase activity as directly isolated in subcellular fractions. Using similar techniques, we also studied the reactivity of a specific PTPase, PTP1B, by immunoprecipitation. This approach provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu that more closely reflects their endogenous reactivity and their potential effect on signal transduction pathways involving reversible protein-tyrosine phosphorylation.PRINCIPAL FINDINGS1. Cellular PTPase activity from 3T3-L1 adipocytes is reversible to a variable extent after oxidative inhibition by exposure to airThe effect of air exposure on PTPases isolated from differentiated murine 3T3-L1 adipocytes was ev...

The BCR/ABL Tyrosine Kinase Induces Production of Reactive Oxygen Species in Hematopoietic Cells

The BCR/ABL oncogene causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and myeloid cells. It is shown here that transformation of the hematopoietic cell lines Ba/F3, 32Dcl3, and MO7e with BCR/ABL results in an increase in reactive oxygen species (ROS) compared with quiescent, untransformed cells. The increase in ROS was directly due to BCR/ABL because it was blocked by the ABL-specific tyrosine kinase inhibitor STI571. Oxidative stress through ROS is believed to have many biochemical effects, including the potential ability to inhibit protein-tyrosine phosphatases (PTPases). To understand the significance of increased production of ROS, a model system was established in which hydrogen peroxide (H(2)O(2)) was added to untransformed cells to mimic the increase in ROS induced constitutively by BCR/ABL. H(2)O(2) substantially reduced total cellular PTPase activity to a degree approximately equivalent to that of pervanadate, a well known PTPase inhibitor. Further, stimulation of untransformed cells with H(2)O(2) or pervanadate increased tyrosine phosphorylation of each of the most prominent known substrates of BCR/ABL, including c-ABL, c-CBL, SHC, and SHP-2. Treatment of the BCR/ABL-expressing cell line MO7/p210 with the reducing agents pyrrolidine dithiocarbamate or N-acetylcysteine reduced the accumulation of ROS and also decreased tyrosine phosphorylation of cellular proteins. Further, treatment of MO7e cells with H(2)O(2) or pervanadate increased the tyrosine kinase activity of c-ABL. Drugs that alter ROS metabolism or reactivate PTPases may antagonize BCR/ABL transformation.

Lipopolysaccharide-induced priming of the human neutrophil is not associated with a change in phosphotyrosine phosphatase activity

The activation of the neutrophil respiratory burst is a two-step process involving an initial `priming' phase followed by a `triggering' event. The biochemical mechanisms which underlie these events are yet to be fully elucidated, but the evidence suggests a crucial role for stimulus-induced tyrosine phosphorylation. The enhanced tyrosine phosphorylation observed upon triggering primed cells may reflect an increase in tyrosine kinase activity or a reduction in the levels of the opposing phosphotyrosine phosphatases (PTPases). We have investigated the latter by examining the possibility that lipopolysaccharide (LPS)-induced priming of the neutrophil respiratory burst involves the suppression of cellular PTPase activity. Purified human neutrophils were incubated for 60 min with and without LPS. Priming of the respiratory burst was confirmed by fMet–Leu–Phe-induced cytochrome c reduction. The level of PTPase activity was assessed by dephosphorylation of [ 32P]RR- src peptide as substrate. Pretreatment of human neutrophils with 200 ng/ml LPS induced a 2.9±0.3 (mean±SEM, n=3, P=0.022) fold increase in the fMet–Leu–Phe-triggered respiratory burst. In the same cells, LPS did not induce a significant change in the total cellular PTPase activity (1.02±0.02-fold, mean±SEM, n=3, P=0.63). Similarly, stimulation of neutrophils with fMet–Leu–Phe or phorbol myristate acetate did not significantly affect the cellular PTPase activity ( P=0.94 and 0.68, respectively). Our results suggest that suppression of PTPase activity is not the mechanism underlying the priming and/or triggering of the neutrophil respiratory burst.

Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1.

Somatostatin (SS-14) and its structural analogue SMS 201-995 (SMS) are recognized as physiological inhibitors of multiple organs and tissue functions through specific membrane receptors (sst1-sst5). The effects of SS-14 and SMS in the growth control of the pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were investigated to identify and clarify the intracellular events involved. In PANC-1 cells, SS-14 and SMS caused inhibition of their basal growth, and that stimulated by epidermal growth factor, with a maximal effect at 0.1-1 microM. To understand the inhibitory mechanisms, we investigated the effects of SS-14 and SMS on phosphotyrosine phosphatase (PTPase) activity and, more specifically, that of tyrosine phosphatase SHP-1 (PTP1C). SS-14 and SMS caused significant increases in total cellular PTPase activity, and particularly SHP-1, with maximal activation within 1 min. Inhibition of membrane tyrosine kinase and p42 MAP kinase activities was also observed, in response to SS-14 and SMS. In MIA PaCa-2 cells, SS-14 and SMS were associated with a positive growth response at 1-10 nM, after 4 days of culture in serum-free medium. Total cellular PTPase activity was slightly increased, but SHP-1 activity could not be detected; its absence in this cell line was confirmed by Western blot. Membrane tyrosine kinase activities were significantly increased by SS-14 and SMS at concentrations needed for maximal growth. p44/p42, which are constitutively active in this cell line, and p38 activities were not affected by somatostatin. In conclusion, somatostatin can exert different effects on human pancreatic cancer cell growth, depending upon the presence or absence of SHP-1. This enzyme can play a key role in the control of cell proliferation, and its cellular presence may determine the therapeutic potential of somatostatin in the control of cancer cell growth.

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-alpha (TNF-alpha) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-alpha 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-alpha 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-alpha, 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-alpha on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited.

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.

Regulation of protein tyrosine phosphatases by insulin and insulin-like growth factor I.

In this study, we have examined the effects of insulin and insulin-like growth factor (IGF)-I on protein tyrosine phosphatase (PTPase) activity in rat L6 skeletal muscle cells. Under basal conditions, about 85% of total cellular PTPase activity was associated with the particulate (Triton X-100-soluble) fraction. Incubation of the cells with 100 nM insulin or IGF-I significantly increased particulate PTPase activity (p < 0.005) without altering activity in the supernatant or Triton X-100-insoluble fractions. Dose responses studies suggested that the effect of each hormone was mediated through its own receptor. PTPase activity was regulated by both acute and chronic insulin and IGF-I treatment. Maximal stimulation by both ligands occurred at 32 h and then declined. By using an antibody and a cDNA specific for PTPase1B, we found that the chronic stimulation of PTPase activity was accompanied by enhanced expression of PTPase1B mRNA and protein. Maximal induction of PTPase1B mRNA and protein by insulin and IGF-I occurred at 12 and 24 h, respectively. Based on these data, it can be suggested that ligand-stimulated PTPase activity might oppose tyrosine kinase-mediated insulin or IGF-I signal transmission and thus desensitize cells to long-term action by insulin and IGF-I. However, it is also possible that PTPase act as positive mediators of insulin and IGF-I action.

Role of Intracellular Ca2+ in the Epidermal Growth Factor-Induced Inhibition of Protein Tyrosine Phosphatase Activity in a Breast Cancer Cell Line

Increased protein tyrosyl phosphorylation in response to growth factors has been assumed to be solely due to activation of protein tyrosine kinases (PTKs). We report that total cellular protein tyrosine phosphatase (PTPase) activity declined in MDA-MB 468 breast carcinoma cells exposed to epidermal growth factor (EGF). The PTPase activity decreased with concentration as well as with time of EGF incubation. As EGF induces increases in intracellular Ca2+ concentrations and such changes may result in depression of PTPase activity, we treated cells with the calcium ionophore A 23187. Increases in calcium induced by the ionophore resulted in activation of cellular PTPases as indicated by increased dephosphorylation of tyrosine phosphorylated EGFR by cellular lysates. Thus, both the extracellular ligand EGF and the intracellular messenger Ca2+ were shown to modulate cellular PTPase activity in MDA-MB 468 breast carcinoma cells. However, EGF-induced decreases in PTPase activity cannot be attributed to EGF-induced increases in intracellular Ca2+ levels.

Exogenous phosphotyrosine modulates epidermal growth factor receptor tyrosine phosphorylation.

Protein tyrosine phosphatases (PTPases) are potential tumor suppressor proteins which reverse the effects of protein tyrosine kinases (PTKs). We hypothesized that the induction of PTPase activity by the nutritional agent O-phospho-L-tyrosine (P-Tyr), a broad PTPase substrate, could potentially enhance total cellular PTPase activity and inhibit cell growth. In this study, we report that P-Tyr inhibited the growth of MDA-MB 468 cells in a dose-dependent fashion. P-Tyr incubation increased total cellular PTPase activity in MDA-MB 468 breast carcinoma cells. The increase of PTPase activity, as measured by a standard radioactive assay for PTPases, occurred within 10 min of P-Tyr incubation and was dependent on the concentration and time of incubation with P-Tyr. The increased PTPase activity in P-Tyr treated cells was also evident from a non-isotopic PTPase assay involving the dephosphorylation of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF)-mediated tyrosine phosphorylation of EGFR was decreased in situ in a time- and dose-dependent manner in P-Tyr-treated cells. Orthovanadate (100 microM for 4 h) inhibited this decrease, implicating the role of cellular PTPase in P-Tyr-mediated control of EGFR tyrosine phosphorylation. Further, EGFR kinase activity was found to be decreased in P-Tyr-treated cells. We conclude that P-Tyr may inhibit cell growth by decreasing cellular tyrosine phosphorylation. Both a decrease in activity of the EGFR kinase and increases in PTPase activity may have accounted for the growth inhibiting property of P-Tyr.

Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death.

Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways. Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent protein-tyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Val5]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of p59fyn, p56/p53lyn, p55blk, and p56lck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our findings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiation-induced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.

Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].

Characterization of a membrane‐associated phosphotyrosyl protein phosphatase from the A431 human epidermoid carcinoma cell line

The A431 human epidermoid carcinoma cell line exhibits a 30-100-fold overexpression of the epidermal growth factor (EGF) receptor. We have characterized a membrane-associated phosphotyrosyl-protein phosphatase (PTPase) in these cells since it seemed reasonable that overexpression of the EGF-receptor tyrosine kinase will be matched by high PTPase activity. Indeed, of 12 cell lines tested, the A431 cells had the highest specific PTPase activity. About 70% of the total cellular PTPase activity was found associated with membranes after cell fractionation. The membrane-associated PTPase was hydrophobic as judged by its behaviour in Triton X-114 phase partitioning. High-performance liquid chromatography (HPLC) on a DEAE column revealed a single, homogeneous species of membrane-associated PTPase with an apparent molecular mass of 43 kDa as determined by HPLC on a gel permeation column in the presence of Triton X-100. Comparison of this PTPase with the membrane-associated PTPase activities present in rat spleen and in the human chronic myelogenous leukemia cell line K562 revealed additional species resolvable by DEAE-HPLC. These findings suggest that cells may possess different PTPase activities depending on their growth and differentiation states.