okadaic-acid and pervanadate

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.

Topics: Cell Survival; Enzyme Inhibitors; HL-60 Cells; Humans; Molecular Structure; Nitrophenols; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Vanadates

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA)

Topics: Animals; Buffers; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Chelating Agents; Egtazic Acid; Elapid Venoms; Enzyme Inhibitors; Evoked Potentials; Ionophores; Mice; Motor Endplate; Motor Neurons; Muscle, Skeletal; Neurotransmitter Agents; Nitrendipine; Okadaic Acid; omega-Agatoxin IVA; omega-Conotoxin GVIA; Presynaptic Terminals; Synaptic Transmission; Vanadates

HER4 is a member of the epidermal growth factor receptor family and has an essential function in heart and neural development. Identification of two HER4 isoforms, HER4 JM-a and JM-b, which differ in their extracellular juxtamembrane region and in their susceptibility to cleavage after phorbol ester stimulation, showed that the juxtamembrane region of the receptor is critical for proteolysis. We now demonstrate that phorbol ester and pervanadate are effective stimuli for HER4 JM-a processing and that the HER4 JM-b isoform does not undergo cleavage in response to any of the stimuli studied. We also show that HER4 JM-a is not cleaved in cells lacking the metalloprotease tumor necrosis factor-alpha-converting enzyme (TACE) and that reexpression of TACE in these cells restores constitutive and regulated processing of HER4 JM-a. Moreover, we show that the sequence specific to the HER4 JM-a juxtamembrane region is sufficient to confer susceptibility to phorbol 12-myristate 13-acetate-induced cleavage of the HER2 receptor. In conclusion, we provide evidence that TACE is essential for the regulated shedding of the HER4 JM-a receptor.

Topics: 3T3 Cells; ADAM Proteins; ADAM17 Protein; Animals; Clone Cells; Cloning, Molecular; Colforsin; Disintegrins; Drosophila Proteins; ErbB Receptors; Kinetics; Marine Toxins; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mice, Knockout; Okadaic Acid; Oxazoles; Protein Isoforms; Receptor, ErbB-4; Recombinant Proteins; Substrate Specificity; Tetradecanoylphorbol Acetate; Thapsigargin; Transfection; Tumor Necrosis Factor-alpha; 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

Peroxidation of membrane lipids occurs in many different neurodegenerative conditions including stroke, and Alzheimer's and Parkinson's diseases. Recent findings suggest that lipid peroxidation can promote neuronal death by a mechanism involving production of the toxic aldehyde 4-hydroxy-2,3-nonenal (HNE), which may act by covalently modifying proteins and impairing their function. The transcription factor NF-kappa B can prevent neuronal death in experimental models of neurodegenerative disorders by inducing the expression of anti-apoptotic proteins including Bcl-2 and manganese superoxide dismutase. We now report that HNE selectively suppresses basal and inducible NF-kappa B DNA binding activity in cultured rat cortical neurons. Immunoprecipitation-immunoblot analyses using antibodies against HNE-conjugated proteins and p50 and p65 NF-kappa B subunits indicate that HNE does not directly modify NF-kappa B proteins. Moreover, HNE did not affect NF-kappa B DNA-binding activity when added directly to cytosolic extracts, suggesting that HNE inhibits an upstream component of the NF-kappa B signaling pathway. Inhibition of the survival-promoting NF-kappa B signaling pathway by HNE may contribute to neuronal death under conditions in which membrane lipid peroxidation occurs.

Topics: Aldehydes; Alzheimer Disease; Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Lipid Peroxidation; Nerve Degeneration; Neurons; NF-kappa B; Okadaic Acid; Protein Synthesis Inhibitors; Rats; Stroke; Transcription Factor AP-1; Vanadates

The expression of the p38 subfamily of mitogen-activated protein kinases (MAPKs) was examined in rat islets of Langerhans and pancreatic beta-cell lines, and its involvement in the regulation of insulin secretion was investigated. Rat islets and several rodent beta-cell lines were shown to express p38 MAPK by Western blotting. The cellular stress agents sodium arsenite and hyperosmotic sorbitol significantly stimulated p38 MAPK activity, as did the tyrosine phosphatase inhibitor sodium pervanadate and the serine/threonine phosphatase inhibitor okadaic acid. Increases in p38 MAPK activity were not consistently correlated with increases in insulin secretion, and the dissociation between p38 MAPK activity and the regulation of insulin secretion was further demonstrated in studies using the specific p38 MAPK inhibitor SB203580, which was without significant effect on the stimulation of insulin secretion by glucose, 4beta phorbol myristate acetate and forskolin. These studies indicate that although p38 MAPK is expressed in pancreatic beta-cells and can be activated pharmacologically, its activity can be dissociated from the exocytotic release of insulin from rat islets of Langerhans.

Topics: Animals; Arsenites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cells, Cultured; Colforsin; Enzyme Inhibitors; Glucose; Homeostasis; Imidazoles; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Male; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Pyridines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium Compounds; Sorbitol; Tetradecanoylphorbol Acetate; 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

Selective activators and inhibitors of insulin signaling cascades in mammalian cells were tested for their effects on insulin stimulated steroidogenesis by ovaries of Aedes aegypti. Bovine insulin in the concentration range of 1.7 microM to 85 microM stimulated ecdysteroidogenesis in vitro. Pervanadate, an inhibitor of tyrosine kinase phosphatase, stimulated ecdysteroid production at concentrations of 250 microM to 1 microM. Okidaic acid, a serine/threonine phosphatase inhibitor, stimulated steroidogenesis with an ED50 of 77.39 nM. A selective inhibitor of tyrosine kinase activity, HNMPA-(AM3), inhibited ecdysteroid production with an IC50 of 14.2 microM. Two selective inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002, inhibited ecdysteroid production at low concentrations (IC50 = 1.6 nM and 30 nM, respectively). These concentrations are similar to those inhibiting insulin action in mammalian cells. A selective inhibitor of mitogen-activated protein kinase, PD098059, had no effect on ecdysteroid production even up to 100 microM. Thus, insulin stimulation of ecdysteroid production by ovaries in vitro appears to be controlled by the tyrosine kinase activity of the mosquito insulin receptor and the signaling cascade involving phosphatidylinositol 3-kinase and protein kinase B.

Topics: Aedes; Animals; Cattle; Ecdysteroids; Enzyme Inhibitors; Female; In Vitro Techniques; Insulin; Mitogen-Activated Protein Kinases; Okadaic Acid; Ovary; Phosphatidylinositol 3-Kinases; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Steroids; Vanadates

1. The modulatory effect of 5-hydroxytryptamine (5-HT) on the gamma-aminobutyric acid(A) (GABA(A)) response was investigated in the neurones freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. 2. 5-HT potentiated GABA-induced Cl- current (IGABA) without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor. 3. Alpha-Methyl-5-HT mimicked the potentiation effect of 5-HT on IGABA while ketanserine blocked it. 1-Oleoyl-2-acetyl-glycerol (OAG) potentiated IGABA, and the effect of 5-HT on IGABA was occluded by OAG pretreatment. In the presence of chelerythrine, 5-HT failed to potentiate IGABA, suggesting that protein kinase C (PKC) is involved in the pathway through which the activation of the 5-HT2 receptor potentiates the IGABA. 4. The facilitatory effect of 5-HT on IGABA remained in the presence of BAPTA-AM. LiCl also had no effect on 5-HT-induced potentiation of IGABA. 5. H-89, genistein, okadaic acid and pervanadate all had no effects on 5-HT potentiation of IGABA. Pertussis toxin treatment for 6-8 h did not block the facilitatory effect of 5-HT on IGABA. 6. The present results show that GABA(A) receptor in the rat SDCN could be modulated in situ by 5-HT, one of the major transmitters involved in the supraspinal control of nociception, and that the phosphorylation of GABA(A) receptor by PKC may be sufficient to support such modulation. The results also strongly support the hypothesis that the cotransmission by 5-HT and GABA has an important role in the spinal cord.

Topics: Animals; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; In Vitro Techniques; Isoquinolines; Membrane Potentials; Neurons; Okadaic Acid; Patch-Clamp Techniques; Protein Kinase C; Rats; Rats, Wistar; Receptors, GABA-A; Serotonin; Signal Transduction; Spinal Cord; Sulfonamides; Vanadates

Inhibition of protein tyrosine phosphatases blocks tumor necrosis factor (TNF)-induced growth modulation and NF-kappaB activation, both mediated primarily through the p60 TNF receptor. How inhibition of the phosphatases affects the p60 TNF receptor or the recently described receptor-associated serine/threonine kinase (p60TRAK) is not known. In this report, we show that this inhibition, when induced by pervanadate, caused the tyrosine phosphorylation of the cytoplasmic domain (CD) of the p60 receptor, as revealed by phosphoamino acid analysis. Furthermore, site-directed mutagenesis indicated that pervanadate specifically induced the phosphorylation of tyrosine-331, which is located in the death domain of the TNF receptor, a domain to which p60TRAK binds. This tyrosine residue was also phosphorylated by purified, recombinant pp60Src in vitro. Inhibition of protein tyrosine phosphatases by pervanadate also led to the inactivation of p60TRAK. In contrast, okadaic acid, a specific inhibitor of protein serine/threonine phosphatase, increased p60TRAK activity. Taken together, these results suggest that protein tyrosine phosphatases play an essential role in phosphorylation of the cytoplasmic domain of the TNF receptor and in regulation of the receptor-associated kinase, and this in turn may play a role in TNF-mediated growth modulation and NF-kappaB activation.

Topics: Amino Acid Sequence; Antigens, CD; Binding Sites; Cytoplasm; Enzyme Activation; Enzyme Inhibitors; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Okadaic Acid; Phosphorylation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins pp60(c-src); Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Recombinant Fusion Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tyrosine; Vanadates

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.

Topics: Adenosine Triphosphate; Animals; Cyclic GMP; Enzyme Inhibitors; Eye Proteins; Hydroquinones; Ion Channel Gating; Marine Toxins; Microcystins; Okadaic Acid; Oocytes; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Phenols; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Rod Cell Outer Segment; Staurosporine; Vanadates; Xenopus laevis

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

Stimulation of Ca2+ (and Mn2+) entry in salivary epithelial cells by carbachol, or thapsigargin, is mediated by an, as yet, unknown mechanism that is dependent on the depletion of Ca2+ from intracellular Ca2+ stores. This study assesses the possible role of protein phosphorylation in the regulation of Ca2+ entry in rat parotid gland acinar cells. Treatment of cells with the protein phosphatase inhibitors okadaic acid, calyculin A, and pervanadate induced a dose-dependent inhibition of carbachol and thapsigargin stimulation of Ca2+ and Mn2+ entry. All three inhibitors decreased carbachol stimulation of internal Ca2+ release, which likely accounts for the inhibition of carbachol-stimulated Ca2+ entry. Thapsigargin-induced internal Ca2+ release was not affected by the treatments. Additionally, all three phosphatase inhibitors decreased Mn2+ entry into cells with depleted internal Ca2+ store(s) (achieved by incubation with either carbachol or thapsigargin in Ca2+-free medium). Treatment of cells with phorbol 12-myristate 13-acetate, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, or staurosporine did not affect divalent cation entry into unstimulated cells or thapsigargin treated cells. Importantly, when cells with depleted internal Ca2+ store(s) were pretreated with staurosporine, or K-252a, the inhibition of Ca2+ entry by calyculin A and okadaic acid, but not by pervanadate, was attenuated. Although the effect of pervanadate remains to be clarified, these results demonstrate a role for protein phosphorylation in the regulation of divalent cation influx in rat parotid acinar cells.

Topics: Animals; Calcium; Carbachol; Enzyme Inhibitors; Male; Manganese; Marine Toxins; Okadaic Acid; Oxazoles; Parotid Gland; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Rats; Rats, Wistar; Stimulation, Chemical; Thapsigargin; Vanadates

The Tyr-phosphorylation of the cytoplasmic domain of the major membrane-spanning band 3, rather than the Ser/Thr-phosphorylation of the membrane proteins (spectrin and band 3 itself), might be functionally related to certain morphological changes of human erythrocytes. This view is supported by the following lines of evidence: a) vanadate or its derivative pervanadate (vanadyl hydroperoxide), which markedly increase the Tyr-phosphorylation of band 3 (without practically affecting the Ser/Thr-phosphorylation of spectrin) promotes a crenation of human erythrocytes; b) okadaic acid, which selectively increases the Ser/Thr-phosphorylation of spectrin and other membrane proteins, does not promote any shape change, at least at a level detectable with scanning electron microscopy.

Topics: Anion Exchange Protein 1, Erythrocyte; Erythrocyte Membrane; Ethers, Cyclic; Humans; Membrane Proteins; Microscopy, Electron, Scanning; Okadaic Acid; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphotyrosine; Spectrin; Tyrosine; Vanadates

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Ethers, Cyclic; Glucose; Islets of Langerhans; Isoenzymes; Mitogen-Activated Protein Kinase 1; Okadaic Acid; Phosphorylation; Phosphotyrosine; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Signal Transduction; Tetradecanoylphorbol Acetate; 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

In previous studies, we demonstrated that while okadaic acid stimulates glucose metabolism, it suppresses the bioresponses of insulin itself in rat adipocytes (Shisheva and Shechter, Endocrinology 129: 2279-2288, 1991). Both stimulation and suppression were attributed to okadaic acid-dependent inhibition of protein phosphatases 1 and 2A. We report here that exposure of adipocytes to staurosporine prior to okadaic acid restored insulin-stimulated actions on glucose metabolism. The effect was half-maximal at staurosporine concentrations as low as 70 nM and was fully expressed (80-87% of the control) at 400-500 nM. Similarly, the insulin-like effect of pervanadate, which was also suppressed by okadaic acid, was restored completely with staurosporine pretreatment. Staurosporine was less effective in restoring cell responses inhibited by high concentrations of okadaic acid, or when added to the cells after okadaic acid. Cell resensitization was unique to staurosporine and could not be produced by various agents that reduce cellular protein kinase A- or protein kinase C-dependent phosphorylation, such as phenylisopropyl adenosine (PIA), K-252a and GF 109203X. Staurosporine (400 nM) partially reversed lipolysis induced by okadaic acid but not that induced by beta-adrenergic stimulation. PIA, which antagonized okadaic acid-induced lipolysis to the same extent as staurosporine, was not capable of restoring insulin responses. Further studies aimed at elucidating this reversing effect revealed that staurosporine did not reactivate okadaic acid-inhibited protein phosphatases 1 and 2A in both cellular and cell-free systems. In summary, we report here a unique dynamic system in which insulin and pervanadate bioeffects can be fully suppressed and again re-expressed without reactivation of protein phosphatase 1 or 2A. The precise site for both effects, although still obscure, appears to be downstream from autophosphorylated insulin receptor.

Topics: Adipose Tissue; Alkaloids; Animals; Drug Interactions; Ethers, Cyclic; Gene Expression Regulation; Insulin; Lipolysis; Male; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rats; Rats, Wistar; Staurosporine; Vanadates

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.

Topics: Aluminum Compounds; Animals; Cell Communication; Cell Line; Connexin 43; Cricetinae; Cricetulus; Embryo, Mammalian; Epidermal Growth Factor; Ethers, Cyclic; Fibroblasts; Fluorides; Intercellular Junctions; Iodine Radioisotopes; Kinetics; Lung; Marine Toxins; Mesocricetus; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Tetradecanoylphorbol Acetate; Vanadates; Vanadium Compounds