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)

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.
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PMID:Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. 138 Jan 27

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.
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PMID:Defective protein kinase C-mediated actions in cystic fibrosis neutrophils. 189 35

Individuals with cystic fibrosis have a defect in the CFTR protein, a chloride channel regulated by cAMP-dependent protein kinase (PKA). The majority of the phosphorylation sites of PKA are located in the R domain of CFTR. It has been postulated that this domain may act as a gate for the chloride channel. Of the many possible mechanisms whereby the R domain could gate the channel, including interdomain interactions, charge distribution, or conformational change, we investigated the possibility that phosphorylation leads to conformational changes in the R domain. To test this hypothesis, a protocol for purification of human R domain peptide synthesized in a bacterial expression system was developed. Purified R domain was phosphorylated by PKA, and CD spectra were obtained. As a result of phosphorylation by PKA, a significant spectral change, indicative of a reduction in the alpha-helical content, was found. CD spectra of the R domain of a shark homologue of CFTR indicated similar changes in conformation as a result of phosphorylation by PKA. In contrast, phosphorylation of the human R domain by PKC, which has only a small influence on CFTR channel activity, failed to elicit CD spectral changes, indicating no conformational change comparable to those induced by PKA phosphorylation. These observations provide the first structural characterization of the R domain and suggest that the gating of the CFTR chloride channel by PKA may involve a conformational change in the R domain.
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PMID:Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. 751 14

The discovery of the cystic fibrosis (cF) gene was the first step to evaluate the function of its product, the "cystic fibrosis transmembrane conductance regulator" protein and the regulation of this gene. CFTR is a cAMP-dependent CI(-)-channel, which is defect in cF. In contrast, a second CI(-)-channel in epithelial cells is activated by increasing intracellular Ca++ and fully functional in cF cells. Increasing intracellular Ca++ not only activates the Ca(++)-dependent channel, but also downregulates the CFTR gene expression in the same epithelial cells, suggesting a feedback mechanism. This regulatory pathway is based on a protein kinase, probably protein kinase C. The results of this study are a prerequisite for a gene therapy in cF which demands intimate knowledge of the regulation of the CFTR gene. In addition, these results suggest different therapeutical strategies to circumvent the defect in cF cells, which is discussed in detail.
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PMID:[Mucoviscidosis--3 years after discovery of the gene]. 751 92

The regulation of basolateral Cl- conductance in renal proximal tubule was investigated by microelectrodes. Our results suggest that this conductance can be activated by cAMP, while increase in cell Ca2+ or activation of PKC has no effect. These properties of this conductance also suggest that CFTR may exist in the basolateral membrane of proximal tubule.
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PMID:Activation of the basolateral Cl- conductance by cyclic AMP in rabbit renal proximal tubule S3 segment. 775 30

The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 x 10(-3) sec-1 to 58.7 x 10(-3) sec-1 when AMP-PNP concentration was raised from 0.5 to 5 mM in the presence of 1 mM MgATP and 180 nM protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of Po or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce Po and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed to PKC alone. AMP-PNP increased Po at temperatures above 30 degrees C but did not cause locking, evidence that the stabilizing interactions between domains, which have been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q10 = 9.6) than the closing rate (Q10 = 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.
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PMID:The CFTR chloride channel: nucleotide interactions and temperature-dependent gating. 956 50

Necturus gallbladder epithelium (NGE) expresses a CFTR-like apical Cl- conductance that can be activated by cAMP. Here, we show that extracellular ATP (100 microM), which is known to elevate intracellular Ca2+ and to hyperpolarize cells by stimulating apical and basolateral K+ conductances, also stimulates an apical Cl- conductance (Ga,Cl), however with a much slower time course. The selectivity sequence of Ga,Cl was SCN- > I- > NO3- > Br- > Cl- >> isethionate (ISE-), but SCN- and I- partially blocked it, which is analogous to observations of CFTR Cl- channels. To disclose a possible role for intracellular Ca2+, gallbladders were incubated with the Ca2+ chelator BAPTA/AM or bathed in solutions containing only submicromolar Ca2+ concentrations. BAPTA partially inhibited the Ca(2+)-mediated hyperpolarization, but did not reduce the ATP-dependent activation of Ga,Cl and the latter was also seen in low extracellular Ca2+. On the other hand, the cAMP-antagonist Rp-8-Br-cAMPS strongly inhibited the stimulation of Ga,Cl by ATP (as well as by forskolin), but left the ATP-induced hyperpolarization unchanged. Preincubation with a low concentration of forskolin markedly enhanced the stimulatory effect of ATP, and this effect was not modified by the selective inhibition of protein kinase C. These data suggest the involvement of different signal transduction pathways in the ATP-dependent activation of K+ and Cl- conductances in NGE. The stimulation of the Ga,Cl appears to be mediated by cAMP but not by elevation of intracellular Ca2+.
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PMID:Activation of an apical Cl- conductance by extracellular ATP in Necturus gallbladder is mediated by cAMP and not by [Ca2+]i. 1051 42

To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.
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PMID:Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs. 1055 81

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.
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PMID:Interleukin-1beta regulates CFTR expression in human intestinal T84 cells. 1065 93

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha(1)-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha(1)-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.
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PMID:Differential regulation of Cl- transport proteins by PKC in Calu-3 cells. 1123 15


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