Phosphotyrosine Phosphatase Inhibitor Research Articles

Abnormal Protein Tyrosine Phosphorylation in Fibroblasts from Hyperapobetalipoproteinemia Subjects

The stimulatory effects of three normal human serum basic proteins (BP), BP I (Mr 14,000, pI 9.10), BP II (Mr 27, 500, pI 8.48), and BP III (Mr 55,000, pI 8.73) on cellular triglyceride and cholesterol formation require intact protein-tyrosine kinase phosphorylation (TKP). Here we examined whether there is an abnormality in TKP in cultured fibroblasts from 11 patients with hyperapobetalipoproteinemia (hyperapoB) that manifest two acylation-stimulatory defects, decreased stimulation of triglyceride synthesis by BP I but enhanced formation of cholesterol by BP II. Soluble and insoluble proteins in Triton X-100 extracts were isolated by immunoprecipitation with a monoclonal anti-phosphotyrosine antibody (MAPA) bound to agarose beads and by ultracentrifugation, respectively, from confluent fibroblasts after incubation for 24 h in supplemented serum-free and lipid-free medium (DMEM/F12). Western blots of insoluble proteins showed that group (Gp) II (Mr 36,000-55,000) and Gp III (Mr 14,000-35,000) from hyperapoB cells, grown in DMEM/F12 medium without BP, had significantly decreased reactivity to MAPA. No significant differences in reactivity to MAPA were detected between normal and hyperapoB cells for Gp I (Mr 97-120,000). BP II, but not BP I or BP III, reversed the decreased reactivity of Gp II and Gp III to MAPA in hyperapoB cells. Sodium vanadate, an inhibitor of phosphotyrosine phosphatases, did not reverse the deficiency in TKP or the 50% deficiency in the stimulation of mass triglyceride by BP I in hyperapoB cells. Tyrosine-phosphorylated Erk-2, a mitogen-activated protein kinase, identified as one of the proteins in Gp II, was significantly decreased in hyperapoB cells. These results provide further evidence for abnormal protein TKP in hyperapoB cells and suggest a possible link between atherosclerotic changes in hyperapoB patients and growth factors upstream from mitogen-activated protein kinase.

The regulation by phosphorylation of 'priming' of phospholipase A2 activity in the neutrophil model system, differentiated HL60 cells.

1 Differential HL60 cells have been utilized as a model system to examine the 'priming' of neutrophil phospholipase A2 activity. In control cells activation of phospholipase A2 by a 5 min stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (100 nM) was essentially undetectable. When cells were primed by preincubation with 5 microns cytochalasin B for 5 min arachidonate release, a measure of phospholipase A2 activation, was observed within 20 s. 2 Priming by cytochalasin B did not involve or require a change in intracellular free calcium concentration. 3 Priming was associated with an increase in general protein tyrosine phosphorylation and could also be induced by the receptor tyrosine kinase agonist granulocyte macrophage colony-stimulating factor (GM-CSF, 20 ng ml-1) and be mimicked by treatment with the phosphotyrosine phosphatase inhibitor perhydrovanadate (0.5 mM). However, increase in MAP kinase activity was not involved in the priming process. 4 Western blot analysis demonstrated that phospholipase A2 was phosphorylated in both control and primed cells, but that an increase in the amount of membrane associated enzyme was found in the primed cells. 5 Thus priming appears to be due to membrane association of the phospholipase and this may be regulated by tyrosine kinase activities.

Vanadates form insoluble complexes with histones

Vanadium oxoanions are known to have a variety of physiological effects including insulin-like activity, inhibition of phosphotyrosine phosphatases, as well as direct interactions with a variety of cellular proteins such as microtubules. In this study, vanadate was found to form insoluble complexes with histones, as well as other positively charged proteins, in a concentration dependent fashion. This interaction occurred over a 0.5–10 mM range which corresponds to the concentration range required for many of vanadate's known physiological effects. Results from precipitation experiments using vanadate solutions with or without the yellow-orange decavanadate indicated that the decamer form is primarily responsible for this precipitation. Vanadate was able to selectively precipitate histones from soluble chromatin as well as from a soluble bacterial protein extract to which a low concentration of histones had been added. Vanadate was also able to effectively precipitate histone from solutions as low as 0.006 mg/mL histone. Thus, the selective precipitation of histones and other positively charged proteins by vanadate can be utilized as a tool for protein purification. In addition, this interaction may provide insight into the mechanisms for the physiological effects of vanadate.

The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages.

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.

Erythrocyte thiol status regulates band 3 phosphotyrosine level via oxidation/reduction of band 3-associated phosphotyrosine phosphatase

Oxidative stress-induced tyrosine phosphorylation has been ascribed to activation of phosphotyrosine kinase or to inhibition of phosphotyrosine phosphatase (PTP). We have previously identified a PTP associated with band 3 in the human erythrocyte membrane, a PTP that is normally highly active and prevents the appearance of band 3 phosphotyrosine. Here we show that treatment of erythrocytes with the thiol-oxidizing agent diamide leads to the formation of PTP disulfides (PTP-band 3 mixed disulfides) and inhibition of dephosphorylation, allowing the accumulation of band 3 phosphotyrosine. Upon reduction of the disulfides, the band 3 phosphotyrosine is dephosphorylated. Erythrocyte thiol alkylation by N-ethylmaleimide results in irreversible PTP inhibition and irreversible phosphorylation. The results are consistent with the notion that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP.

Role of Oxidative Stress in the Action of Vanadium Phosphotyrosine Phosphatase Inhibitors: REDOX INDEPENDENT ACTIVATION OF NF-κB

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadium-based compounds. Sodium orthovanadate (Na3VO4), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na3VO4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na3VO4. In contrast, both BMOV and pV(phen) strongly activated NF-kappaB. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-kappaB activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-kappaB by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-kappaB activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.

Modulation of Protein Tyrosine Phosphorylation by the Extracellular Matrix

Fibronectin (FN) cross-linked to fibrin following injury provides the provisional matrix required for cells to begin tissue repair. Our previous work has demonstrated that fibroblasts adherent to multimeric FN within the context of a fibrin matrix (FN–;fibrin) exhibit clear phenotypic differences from those adherent to a dimeric FN-coated surface. We hypothesize that this response to multimeric FN may be mediated by altered protein tyrosine phosphatase activity following integrin activation. Methods: NIH 3T3 cells were plated in the presence or absence of pervanadate (PV), a phosphotyrosine phosphatase inhibitor, on wells coated with FN or FN–;fibrin matrix. Spread cell areas were measured after increasing incubation times and are recorded as mean cell area (mm2) ± SEM. Alternatively, cells were lysed and equal amounts of protein were analyzed by immunoblot using a monoclonal antibody specific for phosphotyrosine. Results: PV significantly inhibited cell spreading on FN–;fibrin matrices. In contrast, PV treatment had little effect on cell area on FN alone. Analysis of cell lysates revealed that protein tyrosine phosphorylation events differ in a substrate-dependent manner. Conclusion: Cell attachment to a FN–;fibrin matrix induces distinct cell shape and cytoskeletal organization. Inactivation of tyrosine-specific phosphatases enhances this distinction and inhibits the spreading of cells attached to this substrate. The phosphotyrosyl protein content of treated cells on FN–;fibrin matrix is also diminished. These results suggest that cell–;extracellular matrix interactions affect the tyrosine phosphorylation balance of the cell, thus modifying cytoskeletal organization and related signaling events.

Regulation of signal transducer and activator of transcription (STAT) 5b activation by the temporal pattern of growth hormone stimulation.

Plasma GH profiles, intermittent in adult male and continuous in adult female rats, respectively, activate unique patterns of gene transcription in male and female rat liver. Pulsatile, but not continuous, GH exposure activates liver STAT5 (signal transducer and activator of transcription-5) by tyrosine phosphorylation, leading to nuclear translocation, and is proposed to play a key role in GH pulse-regulated male-specific liver gene expression. The mechanisms underlying the GH pattern dependence of STAT5 activation are presently investigated using a rat hepatocyte-derived cell line. Rat GH stimulated tyrosine phosphorylation followed by serine or threonine phosphorylation, leading to activation of the DNA-binding activity of STAT5b, the major STAT5 form present in these cells. Maximal STAT5b activation required a full 20 min at a receptor-saturating GH concentration of 50 ng/ml, suggesting that hormone binding leading to receptor dimerization is a relatively slow process. Repeat cycles of GH pulsation led to repeat cycles of STAT5b activation followed by deactivation, similar to rat liver in vivo. Full responsiveness to succeeding GH pulses required a minimum GH off-time of > or = 2.5 h, but was independent of new protein synthesis. Continuous GH exposure led to down-regulation of activated STAT5b, consistent with the desensitization of this GH pulse-activated pathway observed in female rat liver. The rapid deactivation of STAT5b after termination of a GH pulse involved phosphotyrosine dephosphorylation as a key first step and could be blocked by pervanadate, a phosphotyrosine phosphatase inhibitor. Unexpectedly, serine/threonine kinase inhibitors also inhibited STAT5b deactivation. These studies establish that STAT5b is responsive to the temporal pattern of GH stimulation and demonstrate a role for both a tyrosine phosphatase and a serine/threonine kinase in resetting this JAK/STAT signaling apparatus so that it may respond to subsequent rounds of GH pulse activation.

Phosphotyrosine phosphatase activity in the macrophage is enhanced by lipopolysaccharide, tumor necrosis factor α, and granulocyte/macrophage-colony stimulating factor: correlation with priming of the respiratory burst

Tyrosine phosphorylation is now recognised as a key event in the activation of the macrophage respiratory burst. Since vanadate, a phosphotyrosine phosphatase (PTP) inhibitor is able to enhance the respiratory burst, we proposed that agents which prime the macrophage for enhance respiratory burst activity may do so by suppressing cellular PTP activity. The level of PTP activity in murine bone marrow-derived macrophages (BMM) was assessed by the ability of cell lysates to dephosphorylate 32P-labelled RR- src peptide. In contrast to our hypothesis, pretreatment of BMM with bacterial lipopolysaccharide (LPS), tumor necrosis factor α (TNF α) or granulocyte/macrophage-colony stimulating factor (GMCSF), agents which prime for enhanced respiratory burst activity, was found to dramatically increase the level of cellular PTP activity. The time-course for this increase correlated well with the time course of priming by these agents. In addition, colony stimulating factor-1, a cytokine which does not prime the macrophage respiratory burst, did not enhance PTP levels. The physiological relevance of the increased PTP activity was further supported by confirming it was active against endogenous tyrosine phosphorylated substrates. Interestingly, phorbol myristate acetate and zymosan, agents which trigger the macrophage respiratory burst, were found to inhibit the PTP activity of BMM. Our results demonstrate the regulation of cellular PTP activity by priming agents and further highlight the importance of tyrosine phosphorylation and dephosphorylation events in the regulation of macrophage function.

Cyclic changes in the organization of cell adhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells.

In this study we have investigated the relationships between the stimulation of tyrosine-specific protein phosphorylation and the state of assembly of cell-cell and cell-matrix adherens-type junctions. Bovine aortic endothelial (BAE) cells were treated with either the phosphotyrosine phosphatase inhibitor pervanadate or with epidermal growth factor (EGF), and the effect of the treatment on the organization of cell contacts and the actin cytoskeleton was evaluated by digital immunomicroscopy. We show here that pervanadate induced a dramatic (about 40-fold) increase in the level of phosphotyrosine labeling of cell-cell junctions, which reached maximal values following 20 minutes of incubation. Concomitantly, the junctional levels of vinculin, actin and plakoglobin increased, followed by a slower recruitment of cadherins to these sites. Upon longer incubation cell-cell junctions deteriorated and stress fibers and focal adhesions were formed. EGF stimulation of serum-starved BAE cells induced a rapid 'wave' of junctional tyrosine phosphorylation, followed by cyclic changes in the local levels of phosphotyrosine labeling. Periodic changes were also found in the intensity of labeling of junctional actin, vinculin and cadherins. These results suggest that tyrosine phosphorylation and the assembly of cell-cell adherens junctions are interdependent processes, and raise the possibility that the cross-talk between the two is responsible both for the regulation of junction formation and for adhesion-mediated signaling.

Muscarinic receptor-stimulated cytosol-membrane translocation of RhoA

Receptor-mediated phospholipase D activation in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) apparently involves the small G protein RhoA. Here, activation of RhoA was examined by measuring cytosol-membrane translocation, which is a sign of RhoA activation. RhoA translocation was induced by guanosine 5′- O-(3-thio)triphosphate in digitonin-permeabilized HEK cells, and in intact cells by the agonist-activated mAChR and by direct activation of heterotrimeric G proteins. RhoA translocation was also induced by the phosphotyrosine phosphatase inhibitor pervanadate, while the tyrosine kinase inhibitors tyrphostin 23 and genistein inhibited the mAChR-induced RhoA translocation. These data suggest that translocation and thus activation of RhoA by the G protein-coupled m3 mAChR in HEK cells apparently involves a tyrosine kinase-dependent reaction. © 1997 Federation of European Biochemical Societies.

Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells.

In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release.

Multiple Routes and Regulation by Tyrosine Phosphorylation Characterize the ATP-Dependent Transport of 2,4-DinitrophenylS-Glutathione in Inside-Out Vesicles from Human Erythrocytes

ATP-dependent efflux routes for 2,4-dinitrophenylS-glutathione (DNP-SG) were investigated using inside-out vesicles from human erythrocytes. Nonlinear double-reciprocal plots of transport at substrate concentrations ranging from 0.07 to 500 μmindicated that multiple transport routes were operative. Sensitivity to naphthyl glucuronide separated [3H]DNP-SG transport into two low-affinity components which by computer analysis exhibitedKmvalues of 330 and 1400 μm, respectively. At low substrate concentrations, two high-affinity routes were observed. The predominant activity (hMOAT3a) exhibited aKmof 0.18 μm(Vmax= 22 pmol/min/mg protein), whereas the second activity (MOAT3b) had aKmof 0.58 μm(Vmax= 16 pmol/min/mg protein). High-affinity transport of DNP-SG increased substantially (2.5-fold) in vesicles preincubated with genistein or other tyrosine kinase inhibitors. Kinetic analyses in vesicles pretreated with 50 μmgenistein showed that increased transport resulted from the appearance of a new activity (hMOAT3c) with aKmof 0.85 μmand a substantially elevatedVmax(80 pmol/min/mg protein). At varying concentrations of genistein, a progression was observed that was consistent with the conversion of hMOAT3b to hMOAT3a and hMOAT3a to hMOAT3c. Phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, produced an opposite progression. Specificity studies showed that hMOAT3a exhibited the highest affinity for various anionic conjugates, and had a notable binding preference for glutathione disulfide. The relative effectiveness of the various inhibitors was similar for hMOAT3a, hMOAT3b, and hMOAT3c, as well as for a corresponding mMOAT3 activity from L1210 mouse cells. The results show that human erythrocytes contain multiple ATP-dependent efflux systems for DNP-SG and that separation of these systems can be achieved on the basis of substrateKmvalue and inhibitor and activator specificity. High-affinity transport can proceed via three activities which appear to be subforms of a single system with differing levels of tyrosine phosphorylation. Multiple hMOAT3 subforms provide flexibility for extruding various anionic conjugates and may have evolved in erythrocytes to expedite the efflux of GS-SG.

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O2-) and its dismutation product, hydrogen peroxide (H2O2). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1–10 μg/ml) decreased H2O2 yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H2O2 release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O2;- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H2O2 and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O2- and H2O2 generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O2;-and *NO production.

Phenylarsine Oxide Modulates Intracellular Dynamics of Alkaline Phosphatase-Containing Granules in Human Neutrophils Stimulated with Phorbol Myristate Acetate or IgG-coated Latex Beads.

We studied the intracellular dynamics of alkaline phosphatase (ALPase) -containing granules in human neutrophils exposed to a phosphotyrosine phosphatase inhibitor, phenylarsine oxide (PAO). Unstimulated neutrophils showed ALPase activity in short rod-shaped intracellular compartments distributed dispersedly throughout the cytoplasm. Enzyme activity was observed in elongated tubular structures and vacuoles in cells stimulated with a protein kinase C activator, phorbol myristate acetate (PMA). When cells were exposed to PAO followed by stimulation with PMA, the enzyme activity was visualized in intracellular compartments, localized in the pericytoplasm, which were associated with the plasma membrane. A tyrosine kinase inhibitor, erbstatin, prevented this re-location of ALPase-containing granules. PAO also prevented the association of ALPase-containing granules with the limiting membrane of phagosomes containing IgG-coated latex beads. Biochemical studies demonstrated that the up-regulation of ALPase activity was not affected by exposure to PAO in cells stimulated with PMA, but was inhibited by erbstatin in cells exposed to PAO followed by stimulation with PMA. The present findings indicate that both tyrosine phosphorylation and phosphotyrosine dephosphorylation are involved in the intracellular dynamics of ALPase-containing granules, and demonstrate that the dynamical alterations of these granules are affected by different stimuli such as PMA or IgG-coated latex beads.

Luteinizing hormone-releasing hormone agonist triptorelin antagonizes signal transduction and mitogenic activity of epidermal growth factor in human ovarian and endometrial cancer cell lines

This study was designed to elucidate the signal transduction mechanisms, mediating the antiproliferative effects of analogs of luteinizing hormone releasing hormone (LHRH) on cell lines derived from human cancers of the ovary (EFO-21, EFO-27) and the endometrium (HEC-1A, Ishikawa). The LHRH agonist triptorelin had no measurable effects on the activity of phospholipase C, protein kinase C, or adenylate cyclase in all 4 cell lines, though these enzymes could be activated through pharmacological stimuli. The proliferation of EFO-21, EFO-27 and HEC-1A cells in serum/phenol red-free medium was significantly stimulated by epidermal growth factor (EGF). This mitogenic effect of EGF was dose dependently antagonized by triptorelin, without affecting the concentrations of EGF receptors. Net tyrosine phosphorylation induced by 1 nM EGF was nearly completely suppressed by simultaneous addition of 10 mu M triptorelin or preincubation for 48 h with 100 nM triptorelin. This inhibitory effect of the LHRH agonist on EGF-induced net tyrosine phosphorylation was partly antagonized by exposure to 100 mu M sodium vandate, an inhibitor of phosphotyrosine phosphatase. In EFO-21, EFO-27, and HEC-1A cells exposure to 100 nM EGF for 5 min induced an approximately 5-fold increase in activity of mitogen activated protein kinase (MAP-kinase)/extracellular signal regulated kinase (ERK) which was virtually nullified, when the cells were exposed for 15 min to 10 mu M triptorelin. These data suggest that LHRH signal transduction mechanisms based on the activation of phospholipase C, protein kinase C, and adenylate cyclase, which operate in the pituitary gonadotroph, are not necessarily involved in the mediation of the antiproliferative effects of triptorelin in these ovarian and endometrial cancer cell lines. Instead our findings support the hypothesis that triptorelin interferes with mitogenic signal transduction, probably through antagonizing tyrosine kinase activity of the EGF receptor.

CAMP Counter-regulates Insulin-mediated Protein Phosphatase-2A Inactivation in Rat Skeletal Muscle Cells

In this study, we examined the mechanism of recently reported inactivation of protein phosphatase-2A (PP-2A) by insulin (Srinivasan, M., and Begum, N. (1994) J. Biol. Chem. 269, 12514-12520) and its counter-regulation by cAMP agonists. Exposure of L6 myotubes to insulin resulted in a rapid inhibition of PP-2A that was accompanied by a 3-fold increase in the phosphotyrosine content of the immunoprecipitated PP-2A catalytic subunit. Pretreatment with (Sp)-cAMP, a cAMP agonist, completely blocked insulin-mediated inhibition of PP-2A activity and decreased the tyrosine phosphorylation of PP-2A catalytic subunit to control levels. To understand the mechanism of counter-regulation of PP-2A by (Sp)-cAMP, cells were pretreated with sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases. Vanadate prevented the effect of (Sp)-cAMP on PP-2A activity and increased the phosphorylation status of PP-2A catalytic subunit to the level observed with insulin. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, an inhibitor of 70-kDa S6 kinase activation, prevented insulin-mediated inactivation of PP-2A, suggesting that these pathways may participate in insulin-mediated phosphorylation and inactivation of PP-2A. These results show that insulin signaling results in a rapid inactivation of PP-2A by increased tyrosine phosphorylation and cAMP agonists counter-regulate insulin's effect on PP-2A by decreasing phosphorylation, presumably via an activated phosphatase.

Intracellular CD22 rapidly moves to the cell surface in a tyrosine kinase-dependent manner following antigen receptor stimulation.

CD22 is a key accessory molecule for Ag receptor signaling in B cells that becomes tyrosine phosphorylated in the signaling process. CD22 associates with sIg and strongly amplifies sIg-induced signals. During B cell development, CD22 is initially expressed intracellularly, with surface expression appearing with IgD expression. We used confocal laser-scanning microscopy and flow cytometry to analyze CD22 translocation responses to signaling events. Cross-linking surface IgM (sIgM) induced rapid movement of CD22 to the cell surface in both Ramos and Daudi B cells, with a 50 to 100% increase in surface expression observed within 5 min of stimulation. The increase in CD22 surface expression was specific in that CD19 expression was not affected. Both cell surface and intracellular CD22 were directed toward the site of sIgM stimulation. Treatment with the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV) also increased CD22 surface expression. The tyrosine kinase inhibitor tyrphostin A10 inhibited CD22 movement at concentrations that inhibited tyrosine phosphorylation of CD22 and other cellular proteins. In contrast to the B cell lines, mature peripheral blood B cells contained very little intracellular CD22 and showed no significant increase in surface expression following sIgM stimulation. The rapid directed movement of intracellular CD22 provides a new mechanism to dynamically regulate Ag receptor signaling, as well as CD22-mediated adhesion.

Effects of mitogens on androgen receptor-mediated transactivation.

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.

Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D.

Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H2O2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H2O2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H2O2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 microM vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H2O2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H2O2-induced PLD activation in vascular endothelial cells.