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)

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.
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PMID:Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate. 799 31

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

Activation of cAMP-dependent protein kinase (cAPK) or protein kinase C (PKC) causes a rapid desensitization of beta 2-adrenergic receptor (beta AR) stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the beta AR, RRSSK263. To determine the role of the individual serines in the cAPK- and PKC-mediated desensitizations, wild type (WT) and mutant beta ARs containing the substitutions, Ser261-->Ala, Ser262-->Ala, Ser262-->Asp, and Ser261/262-->Ala, were constructed and stably transfected into L cells. Results showed that serine 262 was the primary site of the cAPK-induced desensitization, whereas either serine 261 or serine 262 was sufficient to confer the 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)/PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization (6-8-fold increase in the EC50) which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser262-->Ala, was partially uncoupled. The Ser262-->Asp mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor. The beta AR expression level played a critical role in determining the pattern of beta AR desensitization; i.e. while the overall desensitization was unaltered within a large range of beta AR expression level (10-300 fmol/mg), the increase in EC50 and decrease in Vmax were differentially affected by the change in the receptor level.
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PMID:cAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase. 808 4

Protein phosphorylation is a key regulatory mechanism for several functions. Although the complex control of organogenesis and growth most likely includes such mechanisms, few reports have examined protein phosphorylation in the developing mammal. The identification and characterization of mammalian embryonic phosphoproteins will allow a greater understanding of the regulation and mechanisms of developmental processes. Phosphorylation of the endogenous mouse proteins during development revealed a 100-kDa protein, located in the cytosolic fraction, to be the major substrate. The Ca(2+)-calmodulin kinase inhibitors, trifluorperazine and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, inhibited this phosphorylation. Inhibitors of protein kinase C (H-7)- and cAMP-dependent protein kinase, as well as the tyrosine kinase inhibitor, genistein, had no effect. One- and two-dimensional phosphoamino acid analysis indicated that phosphothreonine was the major phosphorylated amino acid. To determine the identity of this protein, the 100-kDa band was isolated and submitted for amino acid analysis and N-terminal sequencing. The N-terminal sequence Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, was identical to the N-terminal sequence of human, hamster and rat elongation factor 2 (EF-2). Western blotting analysis confirmed that the 100 kDa protein was EF-2. Our results of phosphorylated EF-2 in the developing mouse are in agreement with those reported in the avian embryo. However, our results differ in that phosphotyrosine detected in avian embryos could not be detected in murine embryos. This is the first report to demonstrate EF-2 in the developing mammalian embryo and its specific phosphorylation pattern. Our data suggest that the functional phosphoregulation of elongation factor 2 during protein synthesis in mammals is conserved from the developing embryo to the adult and thus emphasizes the importance of EF-2 in normal development and survival.
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PMID:Identification of a 100-kDa phosphoprotein in developing murine embryos as elongation factor 2. 811 99

Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+]i.
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PMID:Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C. 819 49

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.
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PMID:Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements. 820 Sep 92

Cardiac ventricular myocytes from several species, including the guinea pig, possess a cAMP-dependent protein kinase A (PKA)-activated Cl- channel. In the present study, the properties of a protein kinase C (PKC)-activated Cl- current were studied in isolated guinea pig ventricular myocytes using the whole-cell arrangement of the patch-clamp technique. Intracellular dialysis of ventricular cells with PKC resulted in the activation of a large background current that displayed time-independent kinetics. In the presence of 146 mmol/L external Cl- and 71 mmol/L internal Cl-, the reversal potential (Erev) of the background current (-17 +/- 1 mV) was close to that of the Cl- equilibrium potential (-18 mV), and the current versus voltage relation for the current was outward rectifying in shape. When [Cl-]i or [Cl-]o was reduced by substitution of Cl- with aspartic acid, Erev for the background current shifted in a manner expected for a Cl(-)-selective channel. Based on Erev measurements, the permeability sequence for this PKC-activated Cl- channel was determined to be SCN- > I- > Br- congruent to Cl-. The PKC-activated Cl- current was not inhibited by the Cl- channel blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (100 mumol/L) but could be blocked by anthracene-9-carboxylic acid (1 mmol/L). Activation of the current was abolished in the presence of the PKC inhibitor staurosporine (2.5 mumol/L). Under conditions designed to cause a maximal activation of the Cl- channels by PKC, the addition of forskolin (1 mumol/L) to stimulate PKA caused only a slight further increase in the amplitude of the Cl- current. Thus, PKC activates a Cl- channel in guinea pig ventricular cells with properties similar but not identical to the PKA-activated channel.
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PMID:Properties of a protein kinase C-activated chloride current in guinea pig ventricular myocytes. 826 85

To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.
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PMID:Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C. 836 Jan 54

We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-beta (PKC-beta), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P.W., Galligan, M.A., Terpening, C.M., Haussler, C.A., Samuels, D.S., Shimizu, Y., Shimizu, N., and Haussler, M.R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-beta, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineral-ocorticoid, and androgen receptors, eliminated PKC-beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.
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PMID:Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. 839 65

Glomerular accumulation of extracellular matrix in diabetes is a potential regulator of mesangial cell-matrix interactions through transmembrane matrix receptors. We now provide evidence that PG production from rat glomerular mesangial cells is increased by Fn. An increase in PG (measured as PGE) was demonstrated in mesangial cell-enriched glomerular cores after 1-h exposure (149 +/- 8% of timed control) and was sustained over a 24-h period (214 +/- 7%). Increased PG production followed exposure to a chymotryptic fragment (120,000 M(r)) of Fn and occurred concomitant with an increase in particulate PKC activity. A tetrapeptide (Arg-Gly-Asp-Ser) with the Arg-Gly-Asp sequence, contained in Fn and the chymotryptic fragment and recognized by specific membrane receptors (integrin matrix-binding proteins), also raised PG levels. As has been shown previously, exposure to high glucose concentration can increase mesangial cell PGE production (from 677 +/- 61 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 1561 +/- 132 pg.mg protein-1.2 h-1 at 50 mM glucose, P < 0.001). The response to the chymotryptic fragment of Fn also was enhanced by concurrent exposure to high glucose concentration (from 2560 +/- 199 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 4672 +/- 358 pg.mg protein-1.2 h at 50 mM glucose, P < 0.001). Coincubation with H-7, an inhibitor of PKC, abolished the PG response to glucose and the chymotryptic fragment. Involvement of PKC was supported further by abrogation of the effect of chymotryptic fragment in mesangial cells cultured for a prior prolonged period with phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fibronectin-induced increase in mesangial cell prostaglandin release. Effect of hyperglycemia and PKC inhibition. 842 Aug 16


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