Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.
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PMID:Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates. 151 90

The ras gene product (p21) is thought to transduce signals from various growth and differentiation factors. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various factors. Nerve growth factors (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as 20% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal growth factor (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic fibroblast growth factor, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).
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PMID:Nerve growth factor induces rapid accumulation of the GTP-bound form of p21ras in rat pheochromocytoma PC12 cells. 154 49

Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal transduction. Using partially purified M factor we demonstrate that the mat1-Pm gene, which controls entry into meiosis, is transcribed in response to a pheromone signal. Strains mutated in the ras1 gene or in ste6, the fission yeast homologue of Ras protein GDP/GTP exchange factor, are unable to induce transcription of mat1-Pm in response to M factor. Furthermore, an activated ras1val17 mutant exhibits a stronger induction of the mat1-Pm transcript. However, transcription still depends on nitrogen deprivation as well as on the presence of pheromone, showing that activation of the Ras1 protein alone does not substitute for any of these signals. The pat1-114 mutant bypasses the ras1/ste6 checkpoint, suggesting that activation of ras1 contributes to inactivation of the pat1 protein kinase.
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PMID:The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription. 156 51

smg p21 is a member of the ras p21/ras p21-like small GTP-binding protein (G protein) superfamily, having the same putative effector domain as ras p21s. In the preceding report, we showed that smg p21 was a major G protein in bovine aortic smooth muscle membranes. Recently, two different smg p21 cDNA clones, designated smg-21A and -B, were isolated from a bovine brain cDNA library. In the present studies, we resolved the bovine aortic smg p21 fraction into two distinct G protein fractions on hydroxyapatite column chromatography and purified them separately to near homogeneity (22K G1 and -2). Both 22K G1 and -2 were specifically recognized by an anti-smg p21 polyclonal antibody. 22K G1 and -2 were identified as smg p21B and -A, respectively, by peptide map and amino acid sequence analyses. Purified smg p21A and -B showed GDP/GTP-binding and GTPase activities similar to each other. The GTPase activities of smg p21A and -B were equally stimulated by smg p21 GTPase activating protein 1 and -2. Moreover, both smg p21A and -B were phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein. These results indicate that smg p21A and -B coexist in bovine aortic smooth muscle membranes and suggest that smg p21A and -B may serve as intermediates for cyclic AMP actions.
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PMID:The molecular heterogeneity of the smg-21/Krev-1/rap1 proteins, a GTP-binding protein having the same effector domain as ras p21s, in bovine aortic smooth muscle membranes. 164 88

The NADPH-oxidase of human neutrophils can be activated in a cell-free system comprised of plasma membrane, cytosol, and an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). Recently, we showed that diacylglycerol acts synergistically with SDS in the cell-free system to stimulate superoxide generation, with concurrent phosphorylation of a 47-kDa cytosolic protein which is thought to be a component of the oxidase (Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559). We report herein that when undialyzed cytosol is used along with either SDS alone or SDS plus diacylglycerol as activators, adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) both stimulated superoxide generation several fold, yielding about the same maximal velocity. ATP and GTP showed lower levels of stimulation. Stimulation by ATP gamma S and GTP gamma S was nonadditive, and showed a 5-7-fold greater specificity for GTP gamma S. ATP gamma S stimulation was inhibited by the nucleoside diphosphate (NDP) kinase inhibitor UDP. In contrast, when extensively dialyzed cytosol was used, most of the stimulation by ATP gamma S was lost, while most of that by GTP gamma S was retained. Addition of GDP restored the ability of ATP gamma S to stimulate, consistent with NDP kinase-catalyzed formation of GTP gamma S from ATP gamma S plus GDP. This activity was demonstrated directly in both cytosol and plasma membrane. Using undialyzed cytosol, phosphorylation of p47 showed a similar nonspecificity for nucleoside triphosphates, due to NDP kinase activity, but revealed the expected ATP specificity when dialyzed cytosol was used. Neither ATP gamma S nor GTP gamma S were good substrates for protein phosphorylation. Under a variety of conditions, phosphorylation of p47 or other neutrophil proteins failed to correlate with oxidase activation. The present studies indicate that SDS and diacylglycerol stimulation of superoxide generation in the cell-free system is independent of protein kinase C or other protein kinase activity, and suggest a novel role for diacylglycerol in cell regulation.
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PMID:Nucleoside triphosphate requirements for superoxide generation and phosphorylation in a cell-free system from human neutrophils. Sodium dodecyl sulfate and diacylglycerol activate independently of protein kinase C. 165 41

The multiplicity of opioid receptors (mu, delta, kappa) and the limited knowledge of their coupling mechanisms explain why cellular and biochemical changes underlying opioid tolerance/dependence remain poorly understood. Following chronic exposure to opioids, both down- and up-regulation of opioid receptors can occur, depending on the receptor type and/or the central region examined. As these changes generally appear after the tolerance is installed, they are very likely not responsible for it. Instead, opioid tolerance seems to be associated with some uncoupling (probably functional rather than physical) of the opioid receptors from G proteins normally associated with them, therefore resulting in a loss of the capacity of these proteins to exchange GDP for GTP. However, considerable variations might exist in the mechanisms underlying tolerance from one opioid receptor type to another. With regard to dependence, an increase in adenylate cyclase activity, and therefore of cyclic AMP levels and certain protein kinase activities, have been claimed to be responsible for this phenomenon in some cell types. As highly selective opioid agonists and antagonists are now available, experiments with such compounds are expected to yield more informative data on the consequences of the chronic stimulation of a given receptor type. This should contribute to a better understanding of the biochemical and cellular events really responsible for the development of morphine tolerance and dependence.
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PMID:Neurobiological mechanisms of opioid tolerance and dependence. 166 19

We examined the dephosphorylation of p36, a protein of D. discoideum that has previously been shown to be phosphorylated in a GDP-dependent manner (Anschutz et al., 1989). Specific dephosphorylation of p36 was found to occur in cell preparations but the activity responsible was strongly dependent upon the concentration of proteins in those extracts. When preparations were diluted, this activity was no longer detectable and the radiolabeled phosphate incorporated into p36 was stable. In contrast, p36 phosphorylation was seemingly unaffected by this treatment. Under the conditions where endogenous dephosphorylating activity was not detectable, the addition of GDP to the reaction resulted in substantial dephosphorylation of p36. The stimulation of this dephosphorylation process occurred at concentrations of GDP that were distinct from those that led to an increased p36 phosphorylation due to the previously reported stimulation of p36 protein kinase activity. Characterization of the dephosphorylation of p36 indicates that the same enzyme is responsible for the endogenous and GDP-stimulated activities. Additionally, these activities are identical when assayed with p36 that had been phosphorylated with ATP or GTP. In contrast to p36 kinase activity, the dephosphorylation of p36 did not display any developmental changes with respect to its regulatory features.
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PMID:Dual role of GDP in the regulation of the levels of p36 phosphorylation in Dictyostelium discoideum. 166 7

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.
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PMID:G-proteins mediate intestinal chloride channel activation. 170 25

Application of 5-hydroxytryptamine (5HT) induces a slowly depolarizing response in the neurons of Aplysia abdominal ganglion. In voltage-clamped cells, 5HT induced a slow inward current that increased steeply with membrane depolarization from -85 mV showing a negative slope conductance, but never reversed into outward when hyperpolarized beyond the equilibrium potential for K+. The 5HT-induced response was markedly augmented in Ca(2+)-free media, but depressed in Na(+)-free media, and unaffected by a change in external potassium. Intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) significantly depressed the 5HT response in a dose-dependent way. Injection of cholera toxin (CTX) selectively blocked the 5HT-induced response, the effect being irreversible. Neither 3'-deoxyadenosine, an inhibitor of adenylate cyclase, nor H-8, an inhibitor of protein kinase A, depressed the 5HT response. 3-Isobutyl-1-methylxanthine (IBMX) did not augment the 5HT response appreciably. The 5HT responses were not depressed at all during a saturated response to Br-cyclic AMP injected intracellularly. It was concluded that the 5HT response is produced by opening of the voltage-dependent Na(+)-channels with activation of CTX-sensitive G-protein but not necessarily with an increase in intracellular cyclic AMP.
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PMID:A slow voltage-dependent Na(+)-current induced by 5-hydroxytryptamine and the G-protein-coupled activation mechanism in the ganglion cells of Aplysia. 171 63

Elementary K+ currents were recorded at 19 degrees C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K+ channel which prevented Na+ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K+ and between -20 mV and +20 mV) a unitary conductance of 66 +/- 3.9 pS. K+(outw.-rect.) channels have one open and at least two closed states. Open probability and tau open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at -7 mV) was as low as 3 +/- 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mumol/l) without a concomitant change of tau open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg++. The cytoplasmic administration of the catalytic subunit of protein kinase A (120-150 mu/ml) or GTP-gamma-S (100 mumol/l) caused a similar channel activation. GDP-beta-S (100 mumol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K+(outw.-rect.) channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein.
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PMID:Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein. 178 9


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