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)

We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.
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PMID:muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties. 131 19

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the mammalian central nervous system and play major roles in synaptic plasticity, neuronal development, and in some neuropathological conditions. Recent studies have suggested that protein phosphorylation of neuronal glutamate receptors by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) may regulate their function and play a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the receptors and to further examine the sites and mechanisms by which the receptors are modulated, we transiently expressed recombinant glutamate receptors in HEK-293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the proposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current-voltage relation or desensitization kinetics. In other experiments, we have demonstrated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a single alternatively spliced exon in the C-terminal domain. These findings suggest that RNA splicing can regulate NMDA receptor phosphorylation and that, contrary to the previously proposed membrane topology model, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal exon with the PKC phosphorylation sites, our preliminary studies indicate that the NMDA-evoked current is potentiated by intracellular PKC. We are currently examining PKC effects on the NMDA-evoked current responses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are directly phosphorylated and functionally modulated by protein kinases. Moreover, by identifying phosphorylation sites within the receptor proteins, our results provide information about the structure and membrane topology of these receptors.
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PMID:Glutamate receptor modulation by protein phosphorylation. 753 May 47

Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [3H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.
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PMID:Modulation of a recombinant glycine transporter (GLYT1b) by activation of protein kinase C. 759 79

1. Diacylglycerols (DAGs) are common intracellular second messengers produced as a result of activation of phospholipase C. We have examined the direct effects of DAG on currents from cloned voltage-dependent potassium channels. Potassium channels were studied by overexpression of their cRNAs in Xenopus oocytes or of their cDNAs in HEK 293 cells, and macroscopic currents were recorded from inside-out membrane patches. 2. When applied to the intracellular side of the patch, 1,2-dioctanoyl-sn-glycerol (C8:0) (DOG) blocks Shaker IR, Kv1.3, and Kv1.6 channels. This block appears macroscopically as a large speeding of the inactivation rate. Longer carbon chain length DAGs (10 and 12 carbons) are less effective in producing this response. 3. DOG is effective at low concentrations, doubling the apparent inactivation rate at 162 nM, and has a fast time course, with a wash-in and reversal to control each within approximately 30 s. 4. Voltage steps delivered with a two pulse protocol in the presence of DOG indicate that recovery from DOG block is voltage dependent. Recovery occurs quickly (tau = 507 ms) when channels are closed quickly by hyperpolarized (-90 mV) potentials, and occurs slowly (tau = 1.3 s) when channels are closed incompletely by depolarized (-60 mV) potentials. 5. The action of DOG is independent of protein kinase C (PKC) activation, because it does not require ATP, nor is it blocked by staurosporin or by the PKC inhibitor peptide 19-36.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Block of cloned voltage-gated potassium channels by the second messenger diacylglycerol independent of protein kinase C. 766 34

Ca2+ fluxes were examined in HEK 293 cells stably expressing the rat or porcine calcitonin receptors (CTRs). Calcitonin (CT) rapidly increased cytosolic Ca2+ ([Ca2+]i) concentrations in these cells in a manner which was sustained in the presence of extracellular Ca2+ ([Ca2+]e). In cells pretreated with CT, elevation of the [Ca2+]e concentration resulted in a further increase in [Ca2+]i which was concentration-dependent with respect to both the concentration of CT and the increment of [Ca2+]e. Untransfected cells, cells transfected with vector alone, and CTR-transfected cells not treated with CT, were unresponsive to [Ca2+]e. The microsomal Ca(2+)-ATPase inhibitor thapsigargin was able to mimic both the acute [Ca2+]i fluxes and responsiveness to [Ca2+]e mediated by CT in these cells. The CT-induced responsiveness to [Ca2+]e was neither mimicked by, nor affected by, activators of the cAMP or protein kinase C pathways. Treatment of cells with pertussis toxin influenced neither the primary Ca2+ fluxes in response to CT or thapsigargin nor the agonist-induced [Ca2+]e influx. Nifedipine failed to block responses to either CT or thapsigargin. These results lead to the important conclusion that the CTR participates in receptor-activated Ca2+ inflow, in which depletion of intracellular Ca2+ pools leads secondarily to influx of extracellular Ca2+.
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PMID:Calcium inflow in cells transfected with cloned rat and porcine calcitonin receptors. 769 52

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1
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PMID:Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. 778 10

These studies characterize the mechanisms involved in terminating the initial Ca2+ transient stimulated by thyrotropin-releasing hormone (TRH). When TRH was added to GH3 pituitary cells that had been treated with thapsigargin to block any agonist-stimulated increase in [Ca2+]i, TRH caused a decrease in [Ca2+]i. The Ca2+ clearing response was seen in pituitary GH3 cells and in nonexcitable HEK 293 cells transfected with TRH receptor cDNA, was evident at basal or elevated [Ca2+]i, and was mediated by the TRH receptor. The Ca2+ clearing response to TRH was not prevented by thapsigargin, Ca2+ ionophores, nimodipine, or replacement of extracellular Na+ but was inhibited by La3+. La3+ also increased the duration of the TRH-evoked [Ca2+]i transient. TRH-stimulated Ca2+ extrusion was directly demonstrated using extracellular fluo-3 free acid. TRH produced a 5-20-fold increase in Ca2+ efflux that was independent of extracellular Na+ and inhibited by vanadate. TRH stimulation of Ca2+ efflux was not reproduced by phorbol esters or inhibited by down-regulation of protein kinase C or staurosporine. The results suggest that agonist-activated Ca2+ efflux may be a universal component of an agonist-activated Ca2+ response and further suggest that a plasma membrane Ca2+ pump may be an effector for G-protein-coupled receptors linked to Ca2+ mobilization.
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PMID:Thyrotropin-releasing hormone activates Ca2+ efflux. Evidence suggesting that a plasma membrane Ca2+ pump is an effector for a G-protein-coupled Ca(2+)-mobilizing receptor. 798 17

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.
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PMID:Receptor-coupled amyloid precursor protein processing. 823 69

We have characterized the phosphorylation of the glutamate receptor subunit GluR1, using biochemical and electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1 homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in neurons.
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PMID:Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. 866 94

UMR106-06 cells predominantly express the C1a isoform of the rat calcitonin (CT) receptor (CTR). We have compared the homologous regulation of the C1a CTR endogenously expressed in UMR106-06 cells with the cloned C1a CTR in transfected HEK 293 cells, in which expression is driven by a heterologous promoter. It was found that treatment of both cell lines with either salmon CT or human CT reduced the density of cell surface CTR in a dose- and time-dependent manner. However, the magnitude of the response was greater in UMR106-06 cells, and salmon CT was more potent than human CT in both cell lines. Recovery from down-regulation was rapid in transfected cells (< 2 h), but was comparatively delayed in UMR106-06 cells, where less than 70% of receptor-binding capacity had returned by 24 h. In both cell lines, treatment with either agonist increased the basal activity of CT-sensitive adenylate cyclase and caused a time-dependent reduction in the responsiveness of adenylate cyclase to a second challenge with CT. Reduced responsiveness occurred under conditions of minimal loss of CTR from the cell surface, consistent with an uncoupling of the receptor from the signal transduction apparatus. Recovery of CT-sensitive adenylate cyclase was complete in transfected cells by 24 h, but was delayed in UMR106-06 cells, paralleling the slow recovery of receptor binding. CT-induced down-regulation of the CTR was not mimicked by receptor-independent activation of protein kinase A or protein kinase C. However, treatment of cells for 24 h, but not for 4 h, with phorbol ester caused a partial loss of CTR binding in UMR106-06 cells and resulted in an approximately 200% increase in CTR binding in transfected HEK 293 cells. CTR messenger RNA levels, as assessed by reverse transcription-PCR, were not changed by any of the above treatments. These results suggest that CT-induced receptor down-regulation and modulation of the ability of CT to activate adenylate cyclase are inherent properties of the receptor, as they can be recapitulated in an otherwise CTR-naive cell line, in which receptor expression is driven by a heterologous gene promoter. Moreover, and in contrast with CTR regulation in osteoclasts, activation of protein kinase A is insufficient for ligand-induced regulation of the CTR in these nonosteoclastic cell lines, and receptor regulation does not appear to involve altered messenger RNA levels.
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PMID:Homologous regulation of the rat C1a calcitonin receptor (CTR) in nonosteoclastic cells is independent of CTR messenger ribonucleic acid changes and cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation. 889 20


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