Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms.

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.     

Preliminary characterization of phosphotyrosine phosphatase activities in human peripheral blood lymphocytes: Identification of CD45 as a phosphotyrosine phosphatase

A preliminary characterization of the protein phosphotyrosine phosphatase (PT-Pase) activity in human peripheral blood lymphocytes (PBL) has been made using two tyrosine-containing peptides and the epidermal growth factor receptor from A-431 cells as substrates. High PTPase activity with a pH optimum near 7.4 was observed in both the membrane and the cytosolic fractions. At least three distinct fractions with PTPase activity were separated on DEAE cellulose columns, indicating that the enzyme is heterogeneous. Vanadate, molybdate, and salts of zinc, copper, and mercury were all effective enzyme inhibitors, although the inhibition was generally incomplete and showed some variation with the enzyme preparation. The difficulty in completely inhibiting PTPase activity in lymphocytes may help explain the variation between laboratories in studies of tyrosine phosphorylation in these cells. Studies with highly purified T lymphocytes obtained by filtration of PBL through nylon wool columns indicated that the activity is present in T cells. Absorption with agarose containing anti-HLe-I, a mouse monoclonal lgGi antibody specific for the leukocyte-specific surface protein T-200 (CD45), removed up to 40% of the PTPase activity. Enzyme activity was recovered on the immunoadsorbent after extensive washing, confirming that the enzyme was being bound to the beads. Immunoabsorbents containing other mouse lgGi antibodies failed to bind PTPase activity, indicating that the binding to beads with anti-HLe-I antibody is specific. Further characterization of the CD45 and PTPase activities in lymphocytes may provide a better under standing of the role of protein tyrosine phosphorylation in the regulation of proliferation and differentiation in these cells.     

Chemical dissection of the effects of tyrosine phosphorylation of SHP-2

The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.     

Purification and characterization of a human recombinant T-cell protein-tyrosine-phosphatase from a baculovirus expression system.

A 48-kDa human T-cell protein-tyrosine-phosphatase (TC.PTPase) and a truncated form missing an 11-kDa C-terminal segment (TC delta C11.PTPase) were expressed by using the baculovirus system and characterized after extensive purification. The full-length PTPase was restricted to the particulate fraction of the cells from which it could be released by a combination of salt and detergent. The enzyme was entirely specific for phosphotyrosine residues. It displayed a low level of activity toward phosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme (RCML), but was 12 times more active toward phosphorylated myelin basic protein (MBP). By contrast, the 37-kDa form localized in the soluble fraction, and its activity toward RCML was 5 times higher than that observed with MBP. The autophosphorylated cytoplasmic domain of the EGF receptor served as substrate for both enzymes. Limited proteolysis of either protein gave rise to a 33-kDa fragment displaying the substrate specificity of the truncated form. These data lend further support to the view that the C-terminal segment of the T-cell PTPase serves a regulatory function, playing an important role in the localization and substrate specificity of the enzyme.     

The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase.

The signalling mechanism and cellular targets of the AT2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser/Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.     

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.     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol     

Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+)

During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.     

Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor.

Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.     

Chicken anemia virus VP2 is a novel dual specificity protein phosphatase

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V(max) of 14,925 units/mg.min and a K(m) of 18.88 microm. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mm orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mm sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V(max) of 28,600 units/mg.min and a K(m) of 76 microm. Mutagenesis of residue Cys(95) to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.     

Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity.

Incubation of human or sheep platelet crude membranes with xanthine oxidase/hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosphatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity in a time-dependent manner, this inhibition being significant within 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20-45 min after the start of the treatment. The inhibition of PTPase activity in general showed good a correlation with the production of thiobarbituric acid-reactive substances (TBARS). The results with several antioxidants suggest that the inhibition of PTPase activity is related to the generation of alkoxyl and/or peroxyl radicals. Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treatment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. However, the amount of protein thiols was reduced only by treatment with trans-2-nonenal. Fluorescence product formation was always higher with trans-2-nonenal, these products being mainly located in the protein fraction. The results with aldehydes suggest that secondary degraded products of lipid hydroperoxides affect PTPase activity. Kinetic studies of PTPase activity indicated that with all treatments enzyme inhibition is mainly due to a decrease in the Vmax value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/ hypoxanthine in the presence of Fe2+/ADP is a very complex, time-dependent process, and that it is probably related, at least after long periods of peroxidation, to changes in protein thiols and amino groups. We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing importance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular.     

Histochemical staining of protein-tyrosine phosphatase activity in primary human mammary carcinoma: relationship with established prognostic indicators.

Protein-tyrosine phosphatase (PTPase) activity in frozen human mammary primary carcinoma tissue sections has been quantitated using a modified histochemical assay. The improved method features the assay of PTPase activity in 12-microns sections of air-dried unfixed tissues, and the use of [2-(N-morpholino)-ethanesulfonic acid] (MES) buffer to prepare stable reaction solutions. Tissue samples from 53 primary human mammary carcinomas were assayed for PTPase activity, and immunohistochemically stained for c-erbB-2 protein-tyrosine kinase expression. Elevated levels of PTPase activity were found in 68% of the tumors compared with the level of activity found in normal human mammary tissues. PTPase activity was co-localized with pathology definitive for carcinoma. Excessive activity was demonstrated throughout the cell, with high activity evident in the cell cytoplasmic membrane and the nucleus. Coexistence of elevated expression of c-erbB-2 and increased PTPase activity was present in 53% of the tumors. In contrast, 15% displayed low c-erbB-2 expression and high PTPase activity, and 24% displayed high c-erbB-2 expression and low PTPase activity. No statistically significant association was found between increased PTPase activity and either c-erbB-2 overexpression or grade and stage of disease in primary human mammary tumors.     

YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopH_C403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.     

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.     

The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s).

The autophosphorylation, from [gamma-32P]ATP, of insulin and epidermal growth factor receptors in rat liver endosomes peaked at 2-5 min and declined thereafter. When autophosphorylation from either [gamma-32P]ATP or unlabeled ATP was stopped after 5 min by adding excess EDTA +/- ATP, the phosphotyrosine (PY) content of each receptor decreased at 37 degrees C with a t 1/2 of 1.6 min. This was equally so whether the PY content of 32P-labeled receptors was analyzed by autoradiography of KOH-treated gels or by Western blotting with PY antibodies of immunoprecipitated receptors. The dephosphorylation reaction was strictly dependent on the presence of sulfhydryl, was unaffected by the addition of rat liver cytosol, and was temperature-dependent. The phosphotyrosine phosphatase(s) (PTPase(s)) appeared to be tightly anchored to the endosomal membrane, since the dephosphorylation reaction was unaffected by sodium carbonate and 0.6 M KCl treatments. However, treatment with Triton X-100 abolished dephosphorylation, implying an intimate association between the PTPase(s) and its substrate in an intact membrane environment. The powerful insulinomimetic agent pervanadate was the most potent inhibitor (50% inhibition at 1 microM). Increasing the dose of injected ligand augmented the rate of insulin and decreased that of EGF receptor dephosphorylation, respectively. Immunoblotting with specific antibodies failed to identify PTPase 1B or T-cell PTPase in ENs, whereas positive signals were seen in plasma membrane. These studies indicate that the phosphorylation state of receptor tyrosine kinases is dynamically regulated, with dephosphorylation, by closely associated PTPase(s), playing an important role.     

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.     

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.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

Cloning and expression of a yeast protein tyrosine phosphatase.

To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR). Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment. The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases. The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein. This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein. The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1. This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step. The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate. Gene disruption of yeast PTP1 has no visible effect on vegetative growth.