Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calyculin A, the potent inhibitor of type 1 (PP1) and type 2A (PP2A) phosphatases, has been employed in order to investigate the role of endogenously activated PP1/PP2A in the signal-transduction pathway of platelet-activating-factor (PAF)-stimulated platelets. Calyculin A alone caused an increase in protein phosphorylation in unstimulated platelets, with the detection of a number of newly phosphorylated proteins, whereas in PAF-stimulated platelets phosphorylation of the major substrates of protein kinase C and myosin light-chain kinase were no longer transient, but phosphorylation was sustained. PP1/PP2A appear to play a role in Ca2+ homoeostasis, as inhibition of PP1/PP2A caused an inhibition of Ca2+ mobilization and Ca2+ influx through the plasma membrane in PAF-stimulated platelets. The effect of calyculin A on Ca2+ mobilization correlated with the observed inhibition of the production of the signal molecule Ins(1,4,5)P3. The release reaction (which is a Ca(2+)-dependent event) was also inhibited by calyculin A. The results are discussed in relation to the possible role of protein kinase C in mediating the events leading to the effects observed with calyculin A.
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PMID:Role of type 1 and type 2A phosphatases in signal transduction of platelet-activating-factor-stimulated rabbit platelets. 804 99

Cultured rat brain capillary endothelial cells expressed a large 86Rb+ uptake component that was dependent on external Na+ and Cl- and that was inhibited by loop diuretics with unusual pharmacological properties: benzmetanide (IC50 = 1-5 microM) = bumetanide (IC50 = 1-5 microM) > piretanide (IC50 = 3-16 microM) = furosemide (IC50 = 7-11 microM). It was activated 2-fold by endothelin-1 (EC50 = 1 nM) and endothelin-3 (EC50 = 9 nM). The actions of endothelins were prevented by BQ-123 (cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu)) in a competitive manner and with a high affinity, thus indicating the involvement of an atypical BQ-123-sensitive, ETA-like receptor that had a high affinity for endothelin-3. Neither protein kinase C nor Ca(2+)-dependent protein kinases mediated the actions of endothelins. Cotransport activity was increased 4-fold by hyperosmotic cell shrinkage. Basal Na(+)-K(+)-Cl- cotransport activity was partially inhibited by isoproterenol and was unaffected by agents that promoted cGMP formation. Calyculin A, an inhibitor of protein phosphatases, stimulated cotransport activity and potentiated the action of endothelin-1, but not that of cell shrinkage. Basal and stimulated cotransport activities were inhibited by genistein, a protein kinase inhibitor with similar potencies, and by staurosporine, which has different potencies. Finally, endothelin-1-stimulated activity was partially and specifically inhibited by interleukin-1. It is concluded that rat brain capillary endothelial cells express a Na(+)-K(+)-Cl- cotransporter that has unique properties and that is regulated by multiple protein kinase/phosphatase systems. It is a target for low concentrations of endothelins and may play a role in brain-to-blood movements of K+.
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PMID:Na(+)-K(+)-Cl- cotransporter of brain capillary endothelial cells. Properties and regulation by endothelins, hyperosmolar solutions, calyculin A, and interleukin-1. 805 Oct 75

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
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PMID:Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells. 814 46

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.
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PMID:Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A. 851 1

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.
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PMID:Modulation of macrophage scavenger receptor transport by protein phosphorylation. 872 20

The goal was to investigate the role of protein kinases in modulating taurine transporter activity in Xenopus laevis oocytes expressing the mouse retinal Na+/C-/taurine transporter. The currents generated by the taurine transporter were studied with a two-electrode voltage clamp and we recorded the maximal current (Imax), presteady-state charge transfer Q, and membrane capacitance Cm. 8-Br-cAMP, a membrane-permeable activator of the cAMP-dependent protein kinase (PKA), decreased Imax (41%), Q (41%) and Cm (10%). Similarly, 1 microM sn-1,2-dioctanoylglycerol (DOG), an activator of the Ca2+/diacylglycerol-dependent protein kinase (PKC), decreased Imax (56%), Q (37%), and Cm (9%). Calyculin A, a specific inhibitor of protein phosphatases 1 and 2A, also produced effects similar to those of 8-Br-cAMP and DOG, and decreased Imax (64 %), Q (38%), and Cm (10%). We conclude that the taurine transporter is regulated by activators of PKA and PKC, and regulation occurs largely by changes in the number of transporters in the plasma membrane.
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PMID:Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes. 877 55

Amiloride-sensitive, Na(+)-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pHi, but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mM HCO3(-), but not in nominally HCO3(-)-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.
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PMID:Shrinkage-induced activation of the Na+/H+ exchanger in Ehrlich ascites tumor cells: mechanisms involved in the activation and a role for the exchanger in cell volume regulation. 883 21

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.
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PMID:Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases. 887 9

The phospholipase A2 (PLA2) neurotoxin, beta-bungarotoxin (beta-BuTX), presynaptically alters acetylcholine release. We previously found that beta-BuTX inhibits protein phosphorylation in rat brain synaptosomes. This inhibition was not due to the inhibition of ATP synthesis, the action of arachidonic acid (AA) metabolites, or the stimulation of phosphatase activities. A typical PLA2 enzyme from Naja naja atra (N. n. atra) venom also inhibited phosphorylation but with lesser potency than that of beta-BuTX. We now report the effects of beta-BuTX and N. n. atra PLA2 on the activities of protein kinases. Treatments of synaptic plasma membrane or cytosol with N. n. atra PLA2 stimulated the activities of cAMP-dependent kinase, Ca2+/calmodulin-dependent kinase II, and protein kinase C (PKC), whereas beta-BuTX had no effect on these kinases. Calyculin A, a phosphatase-1 and -2A inhibitor, increased the stimulation of phosphorylation by N. n. atra PLA2, indicating that the stimulation is not due to an inhibition of phosphatase activities. The stimulation of PKC by N. n. atra PLA2 appears to be mediated by free fatty acids (FFAs) resulting from phospholipid hydrolysis by PLA2, since (1) treatment of either synaptic plasma membrane or cytosol with N. n. atra PLA2 produced large amounts of FFAs, and (2) AA, an exogenous FFA, stimulated PKC activity to an extent similar to that caused by N. n. atra PLA2. Thus, the mechanisms of action of beta-BuTX and N. n. atra PLA2 appear quite different from each other although both agents inhibit phosphorylation in intact synaptosomes.
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PMID:Differential effects of snake venom phospholipase A2 neurotoxin (beta-bungarotoxin) and enzyme (Naja naja atra) on protein kinases. 893 37

We have recently reported that Ser/Thr phosphatases play a key role in regulating natural killer (NK) cell lytic activity and that calyculin A and okadaic acid affect this activity differently [Bajpai and Brahmi (1994) J. Biol. Chem. 269, 18864-18869]. Here, we investigate a mechanism that might account for this differential action of calyculin A and okadaic acid on NK cells. Calyculin A specifically inhibited the lytic activity of YT-INDY, an NK-like cell line, and hyperphosphorylated 60 and 78 kDa proteins. The kinetics of appearance of these two proteins was correlated with the loss of lytic activity. In contrast, okadaic acid did not significantly affect either of these activities. The 78 kDa protein is localized in the cytosolic compartment whereas the 60 kDa protein is distributed equally between the membrane and the cytosolic fractions. Both proteins display a kinase activity and are phosphorylated mainly at serine and threonine residues but not at tyrosine residues. The activation of these kinases is specific to calyculin A treatment; it is independent of protein kinase C, protein kinase A, Ca2+, phosphotyrosine phosphatase and protein synthesis de novo. In conclusion, we have demonstrated that calyculin A, but not okadaic acid, hyper-phosphorylates two proteins with Ser/Thr kinase activity, thus explaining the differential regulation of NK cells by these two Ser/Thr phosphatase inhibitors.
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PMID:Regulation of natural killer cell-mediated cytotoxicity by serine/threonine phosphatases: identification of a calyculin A-sensitive serine/threonine kinase. 894 80


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