Potent Protein Tyrosine Phosphatase Inhibitor Research Articles

Novel Tyrosine Phosphorylation Signals in the Nucleus and on Mitotic Spindle Fibers and Lysosomes Revealed by Strong Inhibition of Tyrosine Dephosphorylation

Protein-tyrosine phosphorylation is one of the posttranslational modifications and plays critical roles in regulating a wide variety of cellular processes, such as cell proliferation, differentiation, adhesion, migration, survival, and apoptosis. Protein-tyrosine phosphorylation is reversibly regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Strong inhibition of protein-tyrosine phosphatase activities is required to undoubtedly detect tyrosine phosphorylation. Our extremely careful usage of Na3VO4, a potent protein-tyrosine phosphatase inhibitor, has revealed not only the different intracellular trafficking pathways of Src-family tyrosine kinase members but also novel tyrosine phosphorylation signals in the nucleus and on mitotic spindle fibers and lysosomes. Furthermore, despite that the first identified oncogene product v-Src is generally believed to induce transformation through continuous stimulation of proliferation signaling by its strong tyrosine kinase activity, v-Src-driven transformation was found to be caused not by continuous proliferation signaling but by v-Src tyrosine kinase activity-dependent stochastic genome alterations. Here, I summarize our findings regarding novel tyrosine phosphorylation signaling in a spatiotemporal sense and highlight the significance of the roles of tyrosine phosphorylation in transcriptional regulation inside the nucleus and chromosome dynamics.

The PPARβ/δ agonist GW0742 modulates signaling pathways associated with cardiac myocyte growth via a non-genomic redox mechanism.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and appear to have beneficial effects in the cardiovascular system. PPARβ/δ has been shown previously to exert an inhibitory effect on cardiac myocyte hypertrophy in vivo and in vitro although the exact mechanism is not fully clear yet. The principal signaling pathways that have been involved in triggering cardiac hypertrophic response are mitogen-activated protein kinases (MAPKs) and PI3K/Akt cascades. In this study, we sought to evaluate the potential effects evoked by PPARβ/δ activation on signaling pathways that are implicated in cardiac myocyte growth responses. The selective PPARβ/δ agonist GW0742 attenuated ERK1/2 and Akt phosphorylation that was stimulated by growth promoting agonists (phenylephrine, insulin or IGF-1). This effect was not reversed by the specific PPARβ/δ antagonist, GSK0660, but was inhibited by vanadate, a potent protein tyrosine phosphatase inhibitor. In addition, GW0742 prevented the oxidation and inactivation of PTEN supporting further the notion that its inhibitory action on the agonist-induced kinase phosphorylation is mediated by the modulation of phosphatase activity. Furthermore, GW0742 abolished the agonist-induced intracellular generation of reactive oxygen species, independently of PPARβ/δ activation. Our data reveals a new non-genomic mechanism of GW0742, which ameliorates the generation of reactive oxygen species and attenuates ERK1/2 and PI3K/Akt signaling, with implications in the regulation of cardiac hypertrophic response.

ChemInform Abstract: Organocatalytic Multicomponent Reaction for the Acquisition of a Selective Inhibitor of mPTPB, a Virulence Factor of Tuberculosis.

AbstractPyrrole‐based libraries (VII) of potent protein tyrosine phosphatase inhibitors with improved potency and selectivity are synthesized via an organocatalyzed multicomponent Mannich‐type reaction.

Abstract 761: The development of cell permeable Shp2 PTP inhibitors

Abstract Protein tyrosine phosphorylation affects cellular activities that control tumor growth and progression. Protein tyrosine phosphorylation, a key regulatory process of signal transduction pathways, is controlled by the action protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Kinases are now a clinically proven anticancer drug target. The development of PTP inhibitors is, by comparison, a relatively new field. Since PTPs do not require a small molecule cofactor they generally show a low propensity to bind a small molecule inhibitor. Nevertheless there are now examples of highly potent PTP inhibitors that provide promising proof-of-principle that PTPs are druggable. Most PTP inhibitors have a charged or highly polar functional group that mimics the phosphotyrosine residue, that often leads to poor ADME and cell permeability properties. We will report our efforts to address the problems of cell permeability we have encountered in our Shp2 PTP inhibitor program. We have previously reported a series of isatin based Shp2 inhibitors based on a compound (NSC-117199) identified from screen of the NCI Diversity Set. We synthesized >100 analogs of NSC-117199 in our lead optimization effort, which yielded several compounds with approximately 50-fold increase in potency and > 10-fold increase in selectivity (Shp2 versus Shp1). However, these Shp2 PTP inhibitors have either a polar nitro or carboxyl group and have no detectable cellular activity. We will report three approaches to address this problem. The first approach was to prepare neutral ester prodrugs. Several compounds have clearly demonstrable activity against Shp2 in cells. A series of esters has also been designed to also improve the water solubility of the PTP inhibitor. A second approach taken was to further modify the isatin scaffold that does not necessitate the use of a prodrug strategy. We will report the discovery of new isatins related to NSC-117199 that have new modifications that result in cell active Shp2 inhibitors. Finally we will report the discovery and preliminary optimization of new classes of cell active Shp2 inhibitors based on alternative scaffolds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 761.

Inhibitory effects of genistein and resveratrol on guinea pig gallbladder contractility in vitro

To observe and compare the effects of phytoestrogen genistein, resveratrol and 17beta-estradiol on the tonic contraction and the phasic contraction of isolated gallbladder muscle strips and to study the underlying mechanisms. Isolated strips of gallbladder muscle from guinea pigs were suspended in organ baths containing Kreb's solution, and the contractilities of strips were measured before and after incubation with genistein, resveratrol and 17beta-estradiol respectively. Similar to 17beta-estradiol, genistein and resveratrol could dose-dependently inhibit the phasic contractile activities, they decreased the mean contractile amplitude and the contractile frequencies of gallbladder muscle strips, and also produced a marked reduction in resting tone. The blocker of estrogen receptor ICI 182780 failed to alter the inhibitory effects induced by genistein and resveratrol, but potassium bisperoxo (1, 10 phenanthroline) oxovanadate bpV (phen), a potent protein tyrosine phosphatase inhibitor, markedly attenuated the inhibitory effects induced by genistein and resveratrol. In calcium-free Kreb's solution containing 0.01 mmol/L egtazic acid (EGTA), genistein and resveratrol inhibited the first phasic contraction induced by acetylcholine (ACh), but did not affect the second contraction induced by CaCl(2). In addition, genistein, resveratrol and 17beta-estradiol also could reduce the contractile responses of ACh and KCl, and shift their cumulative concentration-response curves rightward. Phytoestrogen genistein and resveratrol can directly inhibit the contractile activity of isolated gallbladder muscle both at rest and in response to stimulation. The mechanisms responsible for the inhibitory effects probably due mainly to inhibition of tyrosine kinase, Ca(2+) influx through potential-dependent calcium channels (PDCs) and Ca(2+) release from sarcoplasmic reticulum (SR), but were not related to the estrogen receptors.

Molecular basis for genistein-induced inhibition of Kir2.3 currents

Inwardly rectifying potassium channels play an important role in the maintenance of membrane potential in neurons and myocardium. Identification of functional regulation mechanisms concerning these channels may lead to the development of specific modulators for these channels. Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. In this study, we have found that among three members of the Kir family (Kir2.3, Kir2.1, and Kir3.4* [a highly active mutant of Kir3.4, Kir3.4-S143T]) we tested, genistein significantly inhibited Kir2.3 currents. Using the two-electrode voltage clamp technique, we have demonstrated that micromole concentrations of genistein concentration-dependently and reversibly inhibited the currents of Kir2.3 channel expressed in Xenopus oocytes with an IC50 of 16.9+/-2.8 microM. Using the whole-cell patch-clamp technique, genistein also inhibited the currents of Kir2.3 channel expressed in HEK293 cells with an IC50 of 19.3+/-3.2 microM. Genistein had little or no effect on Kir2.1 and Kir3.4* currents. The effect of genistein on Kir2.3 currents was not affected by vanadate, a potent protein tyrosine phosphatase inhibitor. Furthermore, the effect of genistein was not mimicked by daidzein, an inactive analogue of genistein, or another potent tyrosine kinase inhibitor, tyrphostin 23. Chimeras between Kir2.3 and Kir2.1 channels were constructed to identify molecular basis that distinguished the effect of genistein on these channels. It was found that the transmembrane domains and the pore region of Kir2.3 channel were important determinant for high sensitivity for genistein inhibition.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

Involvement of phosphatidylcholine hydrolysis by phospholipase C in prostaglandin F2alpha-induced 1,2-diacylglycerol formation in osteoblast-like MC3T3-E1 cells.

We previously demonstrated that a prostaglandin F2alpha (PGF2alpha)-induced, sustained increase in 1,2-diacylglycerol (DAG) production was important for proliferation in osteoblast-like MC3T3-E1 cells. The 1,2-DAG formation is mediated by various enzymes, such as phos-phoinositide (PI)-specific phospholipase C (PLC), phospholipase D (PLD), and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). In the present study, to elucidate the mechanism of the 1,2-DAG formation, we have examined the PGF2alpha-induced production of [(3)H]phosphorylcholine, a product of PC-PLC activity, in [(3)H]choline-labeled MC3T3-E1 cells. The PGF2alpha-induced [(3)H]phosphorylcholine production was inhibited by genistein, a potent protein tyrosine kinase inhibitor, and increased by vanadate, a potent protein tyrosine phosphatase inhibitor. However, there were no effects after treatment with protein kinase C (PKC) inhibitors, the guanosine triphosphate (GTP) binding protein activator, NaF/AlCl(3), a Ca(2+)-ionophore, or the potent activator of PKC, phorbol 12-myristate 13-acetate (PMA), suggesting that a tyrosine kinase(s) was involved in the PGF2alpha-induced [(3)H]phosphorylcholine formation. Furthermore, a PGF2alpha analogue, 16-(3-trifluoromethylphenoxy)-Omega-tetranor-trans-Delta(2) PGF2alpha methyl ester (ONO-995), stimulated the proliferation of MC3T3-E1 cells to a level similar to that seen with PGF2alpha, and also caused phosphorylcholine and 1,2-DAG generation. However, neither an increase in intracellular free calcium ion ([Ca(2+)]i) levels by PI-PLC, nor phosphatidylethanol formation (and choline production) by PC-PLD were observed. From these results, we conclude that PGF2alpha-induced 1,2-DAG accumulation was mediated mainly via tyrosine kinase(s)-dependent PC hydrolysis by PLC activity in osteoblast-like MC3T3-E1 cells.

Hepatitis C Virus and HIV Envelope Proteins Collaboratively Mediate Interleukin-8 Secretion through Activation of p38 MAP Kinase and SHP2 in Hepatocytes

Hepatitis C virus (HCV) infects approximately 40% of human immunodeficiency virus (HIV) patients, and the resulting hepatic dysfunction that occurs is the primary cause of death in patients with co-infection. We hypothesized that hepatocytes exposed to HCV and HIV proteins might be susceptible to injury via an "innocent bystander" mechanism. To assess this, we studied the effects of envelope proteins, E2 of HCV and gp120 of HIV, in model HepG2 cells. Upon co-stimulation with HCV-E2 and HIV-gp120, we observed a potent proinflammatory response with the induction of IL-8. Furthermore, our studies revealed that HCV-E2 and HIV-gp120 act collaboratively to trigger a specific set of downstream signaling pathways that include activation of p38 mitogen-activated protein (MAP) kinase and the tyrosine phosphatase, SHP2. Both specific inhibitors of p38 MAP kinase and sodium vanadate, a potent protein-tyrosine phosphatase inhibitor, blocked IL-8 production in a dose-dependent manner. The role of p38 MAP kinase and SHP2 was further defined by transiently overexpressing dominant negative mutants of these proteins into HepG2 cells. These studies revealed that overexpression of an inactive p38 MAP kinase or SHP2 mutant partially abrogated HCV-E2- and HIV-gp120-induced IL-8 production. Further studies revealed that IL-8 induction was not mediated through activation of the NF-kappa B pathway. However, HCV-E2 plus HIV-gp120 was shown to increase the DNA binding activity of AP-1. These results emphasize that expression of the proinflammatory chemokine IL-8, induced by HCV-E2 and HIV-gp120, may be mediated through p38 MAP kinase and SHP2 in an NF-kappa B-independent manner, albeit through AP-1-driven processes.

Conversion of Ca2+ salt of an organic compound to its Li+ salt to simplify the fast atom bombardment mass spectrum.

The FAB mass spectrum of the Ca(2+) salt of RK-682 (1, MW 368), a potent protein tyrosine phosphatase inhibitor, shows a complex pattern due to Ca(2+) adduct ions with multimers of 1 and their decomposition ions. Addition of LiCl greatly simplified the FAB mass spectrum, providing a prominent Li(+) adduct ion of 1 at m/z 381 [M+2Li-H](+). The addition of LiCl also greatly simplified the FAB mass spectrum of calcium pantothenate. This approach may be generally useful for molecular weight determination of multivalent metal salts of organic compounds, or organic compounds that can form Li salts, by FAB mass spectrometry.

Genistein inhibits cardiac L-type Ca(2+) channel activity by a tyrosine kinase-independent mechanism.

It has been suggested that protein tyrosine kinase (PTK) activity can directly regulate cardiac L-type Ca(2+) channels. This conclusion is based to a large extent on the observation that the PTK inhibitor genistein can inhibit the cardiac L-type Ca(2+) current. The purpose of the present study was to determine whether the ability of genistein to inhibit cardiac L-type Ca(2+) channel activity is due to inhibition of PTK activity. Genistein significantly reduced the magnitude of the L-type Ca(2+) current in guinea pig ventricular myocytes recorded using the whole-cell patch-clamp technique. However, this effect was associated with extracellular, not intracellular, application of the drug. Peroxovanadate (PVN), a potent protein tyrosine phosphatase inhibitor, had no effect on the basal Ca(2+) current. PVN was also ineffective in preventing the inhibitory effect of genistein. Internal perfusion of cells with a pipette solution containing ATPgammaS was used to prevent reversibility of phosphorylation-dependent processes. This treatment did not alter the inhibitory effect of genistein, although it did result in irreversible protein kinase A-dependent regulation of the Ca(2+) current. Bath application of lavendustin A, a PTK inhibitor that is structurally unrelated to genistein, did not affect the Ca(2+) current amplitude. The inhibitory effect of genistein was also associated with a hyperpolarizing shift in the voltage dependence of Ca(2+) channel inactivation. These results are consistent with the conclusion that the cardiac L-type Ca(2+) current is not directly regulated by PTK activity and that the inhibitory effect of genistein is due to direct non-catalytic blockade of the channels.

Asymmetric synthesis of a 3-acyltetronic acid derivative, RK-682, and formation of its calcium salt during silica gel column chromatography.

RK-682 was reported to be a potent protein tyrosine phosphatase inhibitor. We found that (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (1) was easily converted to its calcium salt during column chromatography on Silica gel 60, and this calcium salt was identical to RK-682 originally isolated from a natural source. Here we report details of the asymmetric synthesis of (R)-1 and its conversion to the calcium salt. Fast atom bombardment mass spectrometric (FAB-MS) analysis of the free and calcium salt forms of RK-682 is also reported.

Intercellular Adhesion Molecule-1 (ICAM-1) Gene Expression in Human T Cells Is Regulated by Phosphotyrosyl Phosphatase Activity: INVOLVEMENT OF NF-κB, Ets, AND PALINDROMIC INTERFERON-γ-RESPONSIVE ELEMENT-BINDING SITES

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in adhesion phenomena involved in the immune response. The strength of adhesion has been shown to be modulated by changes in ICAM-1 gene expression. In T cells, signaling pathways are intimately regulated by an equilibrium between protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The use of bis-peroxovanadium (bpV) compounds, a class of potent PTP inhibitors, enabled us to investigate the involvement of phosphotyrosyl phosphatases in the regulation of ICAM-1 gene expression in human T cells. Here, we demonstrate for the first time that inhibition of PTP results in an increase of ICAM-1 surface expression on both human T lymphoid and primary mononuclear cells. The crucial role played by the NF-kappaB-, Ets-, and pIgammaRE-binding sites in bpV[pic]-mediated activation of ICAM-1 was demonstrated using various 5' deletion and site-specific mutants of the ICAM-1 gene promoter driving the luciferase reporter gene. Co-transfection experiments with trans-dominant mutants and electrophoretic mobility shift assays confirmed the importance of constitutive and inducible transcription factors that bind to specific responsive elements in bpV-dependent up-regulation of ICAM-1 surface expression. Altogether, these observations suggest that expression of ICAM-1 in human T cells is regulated by phosphotyrosyl phosphatase activity through NF-kappaB-, Ets-, and STAT-1-dependent signaling pathways.

Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases

Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs.

Evidence for a Tyrosine Kinase-Dependent Activation of the Adenylyl Cyclase/PKA Cascade Downstream from the G-Protein-Linked Endothelin ETA Receptor in Vascular Smooth Muscle

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanced cAMP production (t1/2, 2.2 min; EC50, 89 ± 6.3 nM) and stimulated activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETBreceptor agonist, had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced both cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggesting a similar mode of activation, whereas responses to ET-1 and forskolin were synergistic. These findings indicate that AC and PKA are activatable via a nonreceptor PTK-dependent pathway downstream from the G-protein-linked ETAreceptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedback mechanism regulating ET-1-induced vasoconstriction and/or mitogenesis.

Biomolecular Basis for Cold-Induced Contraction of Coronary Arteries in the Newborn Lamb: Implication in Myocardial Dysfunction after Hypothermic Cardiopulmonary Bypass ♦ 98

Introduction: Despite improved overall outcome after open heart surgery, early myocardial dysfunction remains a problem in the infant and the neonate in the post hypothermic cardiopulmonary bypass recovery period. We hypothesize that a vasoconstriction of the conductance coronary arteries (CA) occurs when exposed to cold, and that this cold induced contraction involves protein tyrosine kinase (PTK)-/protein tyrosine phosphatase (PTP)-dependent signal transduction pathways. Methods: Newborn lamb 3-mm CA ring segments (1mm diameter) were studied in tissue bath (Krebs buffer, 21%O2, 5%CO2) for isometric contraction during a 2-hour exposure to hypothermia (7° & 17°C). In parallel, 8-mm CA ring segments were evaluated for changes in protein tyrosine phosphorylation using standard electrophoretic and immunoblotting techniques (monoclonal anti-phosphotyrosine antibody & chemiluminescence detection). Na-orthovanadate (SOV), a potent PTP inhibitor, and genestein (Gen), a non selective PTK inhibitor, were used separately and in combination to evaluate their effect on contractile behavior and tyrosine phosphorylation of CA during cooling (7° or 17°C) vs. 37°C. Results: Tissue bath cooling to 17°C resulted in sustained contraction (25% KCl, n=4), reversible upon rewarming. CA segments subjected to 7°C cooling demonstrated a transitory contraction at≈17°C, more prominent during rewarming (5-15% KCl). Cold-induced contraction was abolished in the presence of Gen and significantly enhanced(2-fold) in the presence of SOV, the latter being partially inhibited in the presence of Gen. There was increased tyrosine phosphorylation of 25, 30-33 and 100-110 kDa proteins detected at 17°C compared to 37°C (n=4), similarly enhanced by SOV and abolished by Gen.

P75, a Member of the Heat Shock Protein Family, Undergoes Tyrosine Phosphorylation in Response to Oxidative Stress

The combination of H2O2 and vanadate generates aqueous peroxovanadium (pV) species, which are effective cell-permeable oxidants, and potent inhibitors of protein-tyrosine phosphatases. As a result, treatment of intact cells with pV compounds significantly enhances protein Tyr phosphorylation. Here we demonstrate that treatment of intact rat hepatoma Fao cells with pV markedly enhances Tyr phosphorylation of a 75-kDa protein, termed pp75. Amino-terminal sequencing of pp75 revealed that this protein is a member of the 70-75-kDa heat shock protein family, which includes PBP-74, glucose-related protein (GRP)-75, and mortalin. Tyr phosphorylation of pp75 is selective, because other proteins that belong to the heat shock protein 70 family, such as GRP-72, Bip (GRP-78), and HSC-70 fail to undergo Tyr phosphorylation when cells are treated with pV. Our findings suggest that heat shock proteins such as pp75 may undergo tyrosine phosphorylation when intact cells are subjected to oxidative stress induced by pV compounds.

Bombesin, Bradykinin, Vasopressin, and Phorbol Esters Rapidly and Transiently Activate Src Family Tyrosine Kinases in Swiss 3T3 Cells: DISSOCIATION FROM TYROSINE PHOSPHORYLATION OF p125 FOCAL ADHESION KINASE

Treatment of quiescent Swiss 3T3 cells with bombesin induces a rapid (</=40 s) and transient increase in the kinase activity of the Src family of tyrosine kinases, as determined by autophosphorylation in immune complex kinase assays (4.6 +/- 0.2-fold stimulation, n = 44) and phosphorylation of exogenous substrates. Phorbol 12, 13-dibutyrate increased the activity of Src family kinases with similar kinetics but was less effective than bombesin. However, Src family kinase activation by bombesin is not dependent either on protein kinase C or Ca2+. Bombesin stimulation of Src family kinase activity could also be dissociated from p125 focal adhesion kinase tyrosine phosphorylation. Neither treatment with cytochalasin D nor placement of the cells in suspension prevented the stimulation of Src family kinase activity induced by bombesin, but both abolished bombesin-induced tyrosine phosphorylation of p125 focal adhesion kinase. The stimulation of the Src family kinase activity by bombesin was completely prevented by treatment with vanadate, a potent inhibitor of protein-tyrosine phosphatases. Bradykinin and vasopressin also stimulated Src family kinase activity transiently, and this stimulation was also inhibited by vanadate. Our results dissect two separate pathways that lead to protein tyrosine phosphorylation in neuropeptide-stimulated Swiss 3T3 cells.

Synthesis of a difluorophosphonomethyl-containing phosphatase inhibitor designed from the X-ray structure of a PTP1B-bound ligand

Protein-tyrosine phosphatase (PTP) inhibitors are potentially valuable pharmacological tools for studying cellular signal transduction and for therapeutic intervention. Small peptides containing the non-hydrolyzable phosphotyrosyl mimetic difluorophosphonomethyl phenylalanine (F 2Pmp) have been shown to be extremely potent PTP inhibitors, with the fluorines increasing inhibitory potency 1000-fold relative to the unfluorinated species. The high PTP affinity of the phosphonodifluoromethyl pharmacophore has allowed the preparation of small molecule inhibitors containing this moiety, which lack any peptide component. The X-ray structure of one such inhibitor. 2-difluoromethylnaphthylphosphonic acid ( 6) complexed to the catalytic site of PTP1B has recently been solved. Computer assisted molecular modelling of this complex indicates that enhanced binding interactions may result by introduction of hydroxyl functionality onto the naphthalene ring system. Herein is reported the synthesis of one such inhibitor 1,1-difluoro-1-[2-(4-hydroxynaphthalenyl)]methyl¦phosphonic acid ( 7), which is prepared in 12 steps from commercially available 1,3-dihydroxynaphthalene. The synthetic approach relies on selective hydroxyl protection and Pd-catalyzed carbonylation to introduce functionality which is subsequently elaborated into the difluorophosphonate structure. The techniques reported herein may be applicable to the preparation of other PTP inhibitors.

Effects of Vanadate on Meiotic Maturation of Porcine Oocytes in Vitro.

Oocyte maturation is triggered by the activation of maturation promoting factor (MPF). In activation of MPF during meiotic maturation of porcine oocytes, a possible involvement of phosphorylation and subsequent dephosphorylation of a inhibitory tyrosine residue in p34cdc2 was examined using vanadate, a potent protein tyrosine phosphatase inhibitor. Porcine oocytes were cultured in porcine follicular fluid supplemented with pregnant mare's serum gonadotrophin with or without vanadate for 48 h. Vanadate blocked germinal vesicle breakdown (GVBD) almost completely at the concentration of 250 and 500 μM. The addition of an effective dose of vanadate into the maturation medium at 24 or 30 h after the start of maturation culture revealed that the porcine oocytes before GVBD were sensitive to vanadate and had their GVBD blocked, but those that had undergone GVBD were not-sensitive to vanadate and reached to the second metaphase normally. When vanadate was removed after 24 h treatment, GVBD occurred in 64% of oocytes but most of them were arrested at the first metaphase with dispersed chromosomes and abnormal spindles. These results suggest that tyrosine dephosphorylation in p34cdc2 takes place during the first meiosis but not in the second meiosis and that a prolonged exposure to vanadate might deteriorate microtubules function of the oocytes.