pervanadate and oxophenylarsine

At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine receptors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet-internalized ;pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here, we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore, we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases, however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.

Topics: Animals; Arsenicals; Female; Mice; Microscopy, Confocal; Neuromuscular Junction; Protein Transport; Protein Tyrosine Phosphatases; Receptors, Nicotinic; Synapses; Vanadates

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

Topics: Animals; Arsenicals; Blood-Brain Barrier; Blotting, Western; Brain; Cell Communication; Cells, Cultured; Electric Impedance; Electrophoresis; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Immunohistochemistry; Matrix Metalloproteinases; Phosphorylation; Protein Tyrosine Phosphatases; Swine; Tight Junctions; Vanadates

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.

Topics: alpha 1-Antitrypsin; Animals; Arsenicals; Carbohydrate Sequence; Endoplasmic Reticulum; Genetic Variation; Glycoproteins; Humans; Liver Neoplasms, Experimental; Mannosidases; Mice; Molecular Sequence Data; Okadaic Acid; Oligosaccharides; Recombinant Proteins; Sodium Fluoride; Transfection; Tumor Cells, Cultured; Vanadates

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.

Topics: Arsenicals; Bacterial Proteins; Benzoquinones; Cell Membrane Permeability; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Humans; Immunoblotting; Lactams, Macrocyclic; Microscopy, Fluorescence; Okadaic Acid; Phosphorylation; Quinones; Receptor Aggregation; Receptors, IgG; Rifabutin; Streptolysins; Temperature; Time Factors; Tyrosine; U937 Cells; Vanadates

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenocorticotropic Hormone; Animals; Arsenicals; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Male; Molecular Weight; Protein Tyrosine Phosphatases; Rats; Rats, Wistar; Signal Transduction; Subcellular Fractions; Vanadates; Zona Fasciculata

Enhancement of tyrosine phosphorylation in cells by the application of pervanadate, an extremely potent phosphotyrosine phosphatase inhibitor, provokes the rapid metalloprotease-dependent cleavage of ErbB-4, a transmembrane receptor tyrosine kinase. The pervanadate-induced proteolysis occurs in NIH 3T3 cells expressing transfected human ErbB-4 and in several cell lines that express endogenous ErbB-4. One product of this proteolytic event is a membrane-anchored molecule of approximately 80 kDa, which is heavily tyrosine phosphorylated and which possesses tyrosine kinase catalytic activity toward an exogenous substrate in vitro. This response to pervanadate is not dependent on protein kinase C activation, which has previously been demonstrated to also activate ErbB-4 cleavage. Hence, the pervanadate and 12-O-tetradecanoylphorbol-13-acetate-induced proteolytic cleavage of ErbB-4 seem to proceed by different mechanisms, although both require metalloprotease activity. Moreover, pervanadate activation of ErbB-4 cleavage, but not that of 12-O-tetradecanoylphorbol-13-acetate , is blocked by the oxygen radical scavenger pyrrolidine dithiocarbomate. A second phosphotyrosine phosphatase inhibitor, phenylarsine oxide, also stimulates a similar cleavage of ErbB-4 but, unlike pervanadate, is not sensitive to pyrrolidine dithiocarbomate. Last, pervanadate is shown to stimulate the proteolytic cell surface processing of a second and unrelated transmembrane molecule: the precursor for amphiregulin, an epidermal growth factor-related molecule. Amphiregulin cleavage by pervanadate occurred in the absence of a cytoplasmic domain and tyrosine phosphorylation of this substrate.

Topics: Amphiregulin; Animals; Arsenicals; EGF Family of Proteins; Enzyme Inhibitors; ErbB Receptors; Glycoproteins; Growth Substances; Intercellular Signaling Peptides and Proteins; Metalloendopeptidases; Mutation; Peptide Fragments; Phenylalanine; Phosphorylation; Protein Kinase C; Protein Tyrosine Phosphatases; Pyrrolidines; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-4; Receptors, Cell Surface; Thiocarbamates; Thiophenes; Tumor Cells, Cultured; Tyrosine; Vanadates

Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down-modulate the tumor necrosis factor (TNF)-mediated activation of the nuclear transcription factor NF-kappa B in ML-1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF-kappa B, we examined the effect of PTPase inhibitors on the TNF-mediated induction of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Like ML-1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF-kappa B; this effect was completely abolished by co-treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM-1, VCAM-1, and ELAM-1 by TNF in MVEC occurred within 6 h and was also completely down-regulated by PAO in a dose-dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF-dependent NF-kappa B activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis.

Topics: Arsenicals; Cell Adhesion; Cells; Cells, Cultured; Diamide; DNA-Binding Proteins; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Humans; Intercellular Adhesion Molecule-1; Monocytes; NF-kappa B; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Necrosis Factor-alpha; Vanadates; Vascular Cell Adhesion Molecule-1

Vanadate mimics, whereas phenylarsine oxide (PAO) antagonizes, the effects of insulin in rat adipocytes. Both vanadate and PAO are documented inhibitors of protein-phosphotyrosine phosphatases. The relationship between the inhibition of 'inhibitory' PTPase and 'stimulatory' PTPase has been studied here in primary rat adipocytes. Low concentrations of PAO (IC50 = 0.6-2.0 microM) blocked the stimulating effects of insulin, vanadate and pervanadate on hexose uptake and glucose metabolism. Inhibition of isoproterenol-mediating lipolysis by vanadate and insulin was not blocked by PAO. The activating effects of okadaic acid on hexose uptake and glucose metabolism, which occur at points downstream to tyrosine phosphorylation, were also not blocked by PAO. Subsequent studies suggested that the PAO-sensitive PTPase comprises a minute fraction of the total adipocytic PTPase activity. To identify its location we applied procedures involving fractionations and activation of non-receptor adipocytic protein tyrosine kinase by PAO and vanadate in cell free assays. We found that the 'inhibitory' PTPase is exclusively associated with the membrane fraction whereas the 'stimulatory' PTPases are present in both the cytosolic and plasma membrane compartments. We next searched for markers, possibly associated with PAO-dependent desensitization and found that several proteins became phosphorylated on tyrosine moieties in the supernatant of PAO but not in vanadate pretreated adipocytes. In summary, we propose the presence of a minute, plasma membrane associated PTPase in primary rat adipocytes, inhibition of which arrests the activation of glucose metabolism. In contrast, inhibition of all the other cellular adipose PTPases, ultimately activates rather than inhibits these same bioeffects.

Topics: 3-O-Methylglucose; Adipocytes; Animals; Arsenicals; Cells, Cultured; Enzyme Inhibitors; Epididymis; Ethers, Cyclic; Glycerol; Glycolysis; Insulin; Isoproterenol; Kinetics; Lipolysis; Male; Methylglucosides; Okadaic Acid; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Vanadates

The extracellular signal-regulated kinases ERK1 and ERK2 are key mediators of mitogenic signals in most cell types. In fibroblasts, sustained activation and nuclear translocation are mandatory for S-phase induction. The events leading to activation of these kinases are well understood, whereas little is known about the mechanism of their translocation. Using indirect immunofluorescence and biochemical analysis we show that ERK1 can translocate to the nucleus in the absence of activation and phosphorylation by upstream kinases when cells are treated with thiol-modifying chemicals. We propose that these chemicals inactivate a protein contributing to the cytoplasmic localization of ERK1.

Topics: Animals; Arsenicals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Nucleus; Enzyme Activation; Ethylmaleimide; Fluorescent Antibody Technique, Indirect; Microtubules; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Platelet-Derived Growth Factor; Protein Tyrosine Phosphatases; Rats; S Phase; Sulfhydryl Reagents; Tubulin; Tyrosine; Vanadates

Most of the inflammatory and proviral effects of tumor necrosis factor (TNF) are mediated through the activation of the nuclear transcription factor NF-kappa B. How TNF activates NF-kappa B, however, is not well understood. We examined the role of protein phosphatases in the TNF-dependent activation of NF-kappa B. Treatment of human myeloid ML-1a cells with TNF rapidly activated (within 30 min) NF-kappa B; this effect was abolished by treating cells with inhibitors of protein-tyrosine phosphatase (PTPase), including phenylarsine oxide (PAO), pervanadate, and diamide. The inhibition was dependent on the dose and occurred whether added before or at the same time as TNF. PAO also inhibited the activation even when added 15 min after the TNF treatment of cells. In contrast to inhibitors of PTPase, okadaic acid and calyculin A, which block serine-threonine phosphatase, had no effect. The effect of PTPase inhibitors was not due to the modulation of TNF receptors. Since both dithiothreitol and dimercaptopropanol reversed the inhibitory effect of PAO, critical sulfhydryl groups in the PTPase must be involved in NF-kappa B activation by TNF. PTPase inhibitors also blocked NF-kappa B activation induced by phorbol ester, ceramide, and interleukin-1 but not that activated by okadaic acid. The degradation of I kappa B protein, a critical step in NF-kappa B activation, was also abolished by the PTPase inhibitors as revealed by immunoblotting. Thus, overall, we demonstrate that PTPase is involved either directly or indirectly in the pathway leading to the activation of NF-kappa B.

Topics: Adenosine Triphosphate; Arsenicals; Base Sequence; Ceramides; Diamide; DNA-Binding Proteins; Dose-Response Relationship, Drug; Ethers, Cyclic; Humans; I-kappa B Proteins; In Vitro Techniques; Interleukin-1; Marine Toxins; Molecular Sequence Data; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Oligodeoxyribonucleotides; Oxazoles; Protein Tyrosine Phosphatases; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vanadates

We investigated the effects of protein tyrosine phosphatase inhibitors, pervanadate and phenylarsine oxide (PAO), on the beta 1 and beta 3 integrin-mediated cell-substrate interaction using normal diploid human fibroblast. Pervanadate treatment of the cells in suspension state resulted in highly elevated levels of cellular protein tyrosine phosphorylation accompanied by loss of beta 1 integrin-mediated adhesion to substrata (i.e., collagen and laminin). In contrast, beta 3 integrin-mediated adhesion to substrata (i.e., fibronectin and vitronectin) of these cells was less affected. Moreover, pervanadate could reverse beta 1 integrin-dependent adhesion, and cells already adhered on collagen or laminin, but not on fibronectin or vitronectin, came off within 30 min upon pervanadate treatment. These effects are likely to be directly mediated by increased cellular protein tyrosine phosphorylation, because another chemical compound, PAO, which also inhibits protein tyrosine phosphatase through a quite different mechanism, also exhibited the specific deterioration of beta 1 integrin-mediated cell-substrate interaction. Upon treatment with these protein tyrosine phosphatase inhibitors, the well developed actin stress fibers were disrupted resulting in the rounding up of cells on fibronectin and vitronectin substrate though they were still attached to the beta 3 integrin-dependent substrates. Using immunoprecipitation and anti-phosphotyrosine immunoblotting, beta 1 integrin itself was shown not to be tyrosine phosphorylated. These results indicate that affinity regulation of beta 1 and beta 3 integrin is differentially controlled and that the specific regulation of beta 1 integrin is due to certain cellular component(s) whose activity is modulated by tyrosine phosphorylation/dephosphorylation.

Topics: Antibodies, Monoclonal; Antigens, CD; Arsenicals; Cell Adhesion; Enzyme Inhibitors; Fibroblasts; Humans; Integrin beta1; Integrin beta3; Integrins; Phosphorylation; Platelet Membrane Glycoproteins; Tyrosine; Vanadates