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)

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.
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PMID:Cloning of a bovine renal epithelial Na+ channel subunit. 757 94

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.
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PMID:The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway. 980 54

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na(+) channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49-65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (tau) and a magnitude (DeltaI (V)). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na(+) channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased DeltaI(V) in hENaC-expressing oocytes to a plateau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g (Na) was attributed to a decrease of membrane capacitance (C (m)), as well as the specific conductance (g(m)/C(m )). The effects on g(m)/C(m) reached a plateau within 15 min, at approximately 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C(m) was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C(m) were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of g(m ) and by changes of g(m)/C(m), indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.
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PMID:Specific and nonspecific effects of protein kinase C on the epithelial Na (+) channel. 1077 14

In an effort to gain insight into how kinases might regulate epithelial Na(+) channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of <2,000 nA. Inhibitors of protein kinases produced varying effects. Chelerythrine, an inhibitor of PKC, produced a significant inhibition of ENaC current, but calphostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G inhibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENaC currents in both oocytes and in M-1 cells, a model for the collecting duct. Site-directed mutagenesis revealed that the staurosporine effect did not require an intact COOH terminus of either the beta- or gamma-hENaC subunit. However, an intact COOH terminus of the alpha-subunit was required for this effect. These results suggest that an integrated kinase network regulates ENaC activity through an action that requires a portion of the alpha-subunit.
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PMID:Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the alpha-subunit. 1079 79

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.7 +/- 1.0 h, whereas betaENaC decreased in 13.9 +/- 3. 0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na(+) reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both beta and gammaENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na(+) transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of beta and gamma but not alphaENaC protein. These results imply a PKC-dependent, long term decrease in Na(+) reabsorption.
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PMID:Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins. 1082 29

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous immunophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na(+) channels in A6 cells. Channel NP(o) was 0.5 +/- 0.08 (n = 6) during the first 5 min but fell close to zero after 20 min. Application of 1 microM rapamycin reactivated Na(+) channels (NP(o) = 0.47 +/- 0.1; n=6), but 1 microM FK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor, mimicked the rapamycin-induced reactivation in a nonadditive manner. However, rapamycin did not reactivate Na(+) channels if cells were exposed to 1 microM FK-506 before rapamycin. In PKC assays, rapamycin was as effective as the PKC inhibitor; however, epithelial Na(+) channel (ENaC) phosphorylation was low under baseline conditions and was not altered by PKC inhibitors or activators. These results suggest that rapamycin activates Na(+) channels by binding FKBP12 and inhibiting PKC, and, in renal cells, despite binding the same immunophilin, rapamycin and FK-506 activate different intracellular signaling pathways.
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PMID:The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells. 1089 19

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an uptake of extracellular Na(+) that is then exchanged for K(+) via stimulation of Na(+)/K(+)-ATPase. While it was generally assumed that this Na(+) uptake is mediated by the activation of Na(+)/H(+) antiport and Na(+)-K(+)-2Cl(-) symport it could be shown recently that, in addition to these transporters, hypertonic stress also stimulates conductive Na(+) entry. In a quantitative study, it was found that the relative contribution of Na(+) conductance, Na(+)/H(+) antiport, and Na(+)-K(+)-2Cl(-) symport to the initial Na(+) import as well as to the RVI process (at 300 --> 400 mosmol/l) is approximately 4 : 1 : 1. When the osmotic sensitivity of these Na(+) importers was tested (at 300 mosmol/l --> 327, 360, 400, 450 mosmol/l) it became clear that Na(+) conductance is the prominent mechanism of RVI from 360 mosmol/l upwards whereas Na(+)/H(+) antiport is the most sensitive transporter with 65 % of its maximal activation at 327 mosmol/l already. Concerning the intracellular regulation of the Na(+) importers involved in RVI it was found that Na(+) concuctance as well as Na(+)-K(+)-2Cl(-) symport - but not Na(+)/H(+) antiport - are activated via PKC. With respect to the molecular correlate of the volume activated Na(+) conductance it could be shown that it exhibits a rather low affinity to amiloride (IC(50) = 6.0 micromol/l) and an overall sensitivity profile of EIPA > amiloride > benzamil = phenamil that, at first sight, would not speak in favor of a typical epithelial type of Na(+) channel (ENaC). Western-blot analysis and RT-PCR techniques, however, revealed that alpha-, beta-, as well as gamma-ENaC are, in fact, expressed in rat hepatocytes. Moreover, by use of an antisense-DNA based approach it could be shown that at least alpha-ENaC is part of the hypertonicity induced Na(+) conductance.
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PMID:The hypertonicity-induced Na(+) conductance of rat hepatocytes: physiological significance and molecular correlate. 1112 14

It has long been known that Na(+) channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na(+) channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (alpha and gamma), novel (delta, eta, and epsilon), and atypical (iota, lambda, and zeta) groups. Steady-state stimulation of Na(+) transport with aldosterone was accompanied by a specific decrease of PKCalpha protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na(+) transport, also decreased PKCalpha levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCalpha. These effects were not accompanied by changes of PKCalpha mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na(+) transport.
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PMID:Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium. 1222 76

Amiloride-sensitive sodium channel (ENaC) plays an important role in recovery from pulmonary edema. Recently, it has been shown that an activation of protein kinase C (PKC) could affect the mRNA expression of ENaC in rat parotid gland cells and A6 distal nephron epithelial cells. To determine whether an activation of PKC would regulate the mRNA expression or the function of ENaC, we stimulated rat alveolar type II epithelial cells with phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, at a concentration of 100 nM. The mRNA expression of alpha-, beta-, and gamma-ENaC subunits and amiloride-sensitive current were measured. PMA inhibited the mRNA expression of all 3 ENaC subunits (alpha-ENaC: 56.0% +/- 12.1%; beta-ENaC: 62.6% +/- 15.9%; gamma-ENaC: 68.5% +/- 10.6%, respectively) and amiloride-sensitive current (control = 7.0 +/- 1.5 microA/cm(2); PMA = 1.7 +/- 0.9 microA/cm(2)) significantly at 24 hours. On the other hand, 4alpha-phorbol didecanoate 4alpha-PDD, inactive form of PMA, had no inhibitory effect on alpha- and gamma-ENaC expression or amiloride-sensitive current. However, no significant difference was seen in beta-ENaC expression between PMA and 4alpha-PDD. GF 109203X, a wide-range PKC inhibitor, blocked the inhibitory effect of PMA on all ENaC subunits mRNA expression. These results suggest that an activation of PKC may play an important role in the regulation of ENaC mRNA expression and function.
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PMID:The impact of phorbol ester on the regulation of amiloride-sensitive epithelial sodium channel in alveolar type ii epithelial cells. 1239 48

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na(+) channel (ENaC) activity. Activation of PKC reduced relative Na(+) reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, alpha-, beta-, and gamma-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in beta- and gamma-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na(+) reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta- and gamma-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of gamma-ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of gamma-ENaC.
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PMID:Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade. 1254 Mar 65


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