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

Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2(+)-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP beta S ATP gamma S, and App[NH]p were effective. GTP gamma S and GDP beta S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 degrees C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.
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PMID:Ca2(+)-induced secretion by electropermeabilized human neutrophils. The roles of Ca2+, nucleotides and protein kinase C. 232 90

Topically applied acetylcholine elicited a hyperpolarizing (ACh-HP) response in about 2/3 of the principal neurons of rabbit superior cervical ganglia isolated in a culture medium for several days. Membrane changes responsible for the ACh-HP were multiple and varied with the level of membrane potential (Vm): At, or less negative than, resting Vm (about -60 mV), membrane conductance (Gm) was increased; at Vm more negative than -70 mV, Gm either increased, decreased, or remained unchanged. The effect of altering extracellular K+ and Ca2+ concentrations suggest that a Ca2(+)-dependent increase in K+ conductance contributes to ACh-HP; however, drugs reported to block such channels (D-tubocurarine at 30 microM or apamin at 200 nM) shortened duration of the ACh-HP but did not depress peak amplitude. The ACh-HP was depressed by the M2-muscarinic antagonist AF-DX 116 and blocked by an intracellular administration of guanosine-5'-o-(2-thiodiphosphate) (GDP-beta-S) or by preincubation with pertussis toxin. Intracellularly injected inositol-1,4,5-triphosphate (IP3) elicited a hyperpolarization associated with an increase in Gm at any Vms. Activators of protein kinase C applied extracellularly, 1,2-oleoylacetylglycerol (OAG) or phorbol-12,13-dibutyrate (Pb(Bu)2) elicited a hyperpolarization with a decrease in Gm at Vms more negative than -50 mV and a reversal potential close to Ec1. In the presence of Li+, ACh-HP was smaller after a repetitive stimulation with ACh. These results suggest that, in these neurons, ACh can directly activate M2-muscarinic receptors coupled to GTP-binding proteins, which leads to both an increase in Ca2(+)-sensitive K+ conductance mediated by the intracellular messenger IP3 and a decrease in a conductance, possibly Gc1, through another phosphatidylinositide pathway.
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PMID:Acetylcholine elicits metabolically mediated M2-muscarinic hyperpolarization in isolated rabbit sympathetic neurons. 239 36

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.
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PMID:Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components. 249 27

In rabbit platelets, collagen (50 micrograms/ml)- or thrombin (0.5 U/ml)-induced diacylglycerol formation was dose-dependently prevented by phorbol 12-myristate 13-acetate (PMA, 2-50 nM). However, collagen-induced arachidonic acid liberation and lysophosphatidylcholine formation were rather enhanced by PMA, while the thrombin-induced liberation was not. We also demonstrated with saponin-permeabilized platelets that collagen (100 micrograms/ml)-induced arachidonic acid liberation was enhanced by GTP gamma S and inhibited by GDP beta S, both dose-dependently. Since these results lead us to consider that protein kinase C affects a guanine-nucleotide-binding protein (G-protein) to modulate phospholipase A2 and C, we investigated this dual effect of PMA on arachidonic acid liberation and diacylglycerol formation induced by G-protein activator. Addition of GTP gamma S (100 microM) to saponin-permeabilized platelets significantly induced these responses, and PMA (2-10 nM)-pretreatment before the cell permeabilization inhibited diacylglycerol formation and enhanced arachidonic acid liberation and lysophosphatidylcholine formation, dose-dependently. Likewise, PMA (20 nM) had differential effects on the similar NaF (20 mM)-induced responses in intact platelets. Contrarily, 10 nM PMA had no effect on diacylglycerol formation caused by an addition of high concentration of Ca2+ (1 mM) alone after the cell permeabilization, while it still had a potentiating effect on arachidonic acid liberation under the condition. These results suggest that protein kinase C may have a dual regulatory effect on the activation of phospholipase A2 (positive feedback) and phospholipase C (negative feedback), probably through influences on two distinct G-proteins associated separately with these two enzymes.
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PMID:Differential effects of phorbol 12-myristate 13-acetate on GTP gamma S-induced diacylglycerol formation and arachidonic acid liberation in saponin-permeabilized rabbit platelets. 249 43

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.
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PMID:Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. 250 24

Polymerization of microfilaments, one of the responses triggered in neutrophils by stimuli such as the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), involves the conversion of actin from the monomeric to the filamentous form. The exact sequence of events responsible for this conversion remains to be defined, but its susceptibility to inhibition by pertussis toxin provides indirect evidence that GTP-binding proteins (G-proteins) are involved. In this report, electropermeabilized cells were used to obtain more direct evidence of a role for G-proteins in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and flow cytometry were used to monitor the formation of filamentous actin. GTP-gamma-S, a nonhydrolyzable analogue of GTP and aluminum fluoride, which in combination with GDP can activate G-proteins, stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells. fMLP-induced actin polymerization in permeabilized cells was inhibited by pretreatment with GDP-beta-S, an analogue of GDP that stabilizes the inactive form of G-proteins. In contrast, stimulation by phorbol 12-myristate 13-acetate (PMA) was largely unaffected by GDP-3-S. These observations indicate that activation of G-proteins is essential for actin assembly induced by receptor-dependent stimuli such as fMLP. Moreover, GTP-binding proteins do not seem to be required in the late stages of the signalling cascade, i.e. after stimulation of protein kinase C.
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PMID:Actin assembly in electropermeabilized neutrophils: role of G-proteins. 251 Jul 21

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.
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PMID:Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein. 253 Feb 40

Glucagon-like peptide-1(7-36)amide [GLP-1(7-36)amide], probably representing an important incretin, binds to receptors on RINm5F cells resulting in an adenosine 3',5'-cyclic monophosphate increase. Guanine nucleotides (GTP, GTP-gamma-S, GDP-beta-S) decreased the binding of GLP-1(7-36)amide to receptors on RINm5F cell membranes. Further analysis revealed that GTP (10(-4) M) decreased the receptor affinity with an increase of the Kd from 2.5 +/- 0.99 x 10(-10) M to 9.43 +/- 2.16 x 10(-10) M. In cross-linking experiments the amount of labeled peptide linked to receptors was reduced in the presence of GTP (10(-4) M). Further studies investigated the involvement of membrane depolarization or changes in the cytosolic free calcium level in the intracellular signaling of GLP-1(7-36)amide-induced insulin secretion. In contrast to fuel and nonfuel secretagogues, GLP-1(7-36)amide did not cause a depolarization of the membrane potential. This was unaffected by various glucose concentrations (0-20 mM) or by previous cell depolarization by D-glyceraldehyde. Similarly, the cytosolic calcium concentration remained unchanged after addition of GLP-1(7-36)amide (10(-12)-10(-8) M). The effect of guanine nucleotides on binding of GLP-1(7-36)amide indicates that the action of the peptide is mediated by the adenylate cyclase system. GLP-1(7-36)amide binding neither changed the membrane potential nor altered the intracellular calcium concentration, making an involvement of the inositol 1,4,5-trisphosphate pathway or an activation of protein kinase C in the postreceptor signaling after GLP-1(7-36)amide binding unlikely.
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PMID:Signal transmission after GLP-1(7-36)amide binding in RINm5F cells. 255 Nov 82

The antigen receptors on B lymphocytes, membrane forms of immunoglobulins, transduce signals regulating B cell growth and differentiation by activating a phosphoinositide-specific phospholipase C. In this report, we describe our recent work aimed at understanding this process in greater detail. We have shown that a GTP-binding component is a necessary cofactor in the stimulation of phospholipase C by mIgM. This component has a number of properties in common with the G protein family of receptor-effector coupling components seen in the adenylate cyclase and other signaling systems. For example, analogues of GTP that cannot be hydrolyzed stimulated mIgM-triggered phosphoinositide breakdown, and an analogue of GDP that cannot be converted to GTP inhibited the reactions. Furthermore, aluminum fluoride, which activates known G proteins, also stimulates phosphoinositide breakdown. The G protein that appears to link mIgM to phospholipase C is not one of the well characterized G proteins involved in the regulation of adenylate cyclase or cGMP phosphodiesterase (GS, Gi, and transducin), as judged by its insensitivity to two bacterial toxins that modify these G proteins, cholera toxin and pertussis toxin. Interestingly, analysis of pertussis toxin sensitivity indicates that there are at least 2 distinct G proteins that couple receptors to phospholipase C. For example, the G protein required for chemotactic peptide receptor signaling in neutrophils is sensitive to pertussis toxin, in contrast to the phosphoinositide signaling G protein in B cells. We have also begun to explore the mechanisms by which mIgM signal transduction can be modulated. Stimulation of protein kinase C with phorbol esters or synthetic DG was found to inhibit mIgM-triggered phosphoinositide breakdown. This regulation probably represents a feedback inhibition that would occur with DG produced by phosphoinositide breakdown. Alternatively, there appear to be other signaling pathways that generate DG33, and they could possibly inhibit phosphoinositide breakdown via protein kinase C. This could be an important locus of regulation during B cell activation. For example, other signals could increase or decrease the potency of this feedback inhibition, and thereby adjust the sensitivity of the B cell to antigen. Alternatively, other agents could stimulate protein kinase C directly, or could stimulate another protein kinase which can do the same thing in this regard, and thereby make the B cell insensitive to antigen by preventing antigen receptor signaling.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Signal transduction via the B cell antigen receptor: involvement of a G protein and regulation of signaling. 255 95

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein. 255 20


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