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)

Cystic fibrosis (CF) is a frequent autosomal recessive genetic disease. The isolation of the gene at the CF locus assigned to the long arm of chromosome 7 band q 31 and defining description of its protein named CFTR (cystic fibrosis transmembrane conductance regulator) promoted understanding the basic biochemical defect. Brief review of relevant literature demonstrates that glycoprotein CFTR is a chloride channel and is activated by a combination of phosphorylation by protein kinase A and binding of ATP. Most common mutation of CF gene, a deletion of the three nucleotides encoding phenylalanine (Delta F508) results in disturbance of chloride transport through membrane of epithelial cells involved in pathomechanism of CF. The way for gene therapy in CF is open, however therapeutic progress is noted on both pharmacologic arena and on the gene cure front. Recombinant vectors utilizing the adenovirus system with high efficiency of CFTR gene transfer to airway epithelium demonstrated in a rat model look promising. The use of retroviruses for CFTR transfer is also advanced mode of somatic gene therapy. An alternative approach suggesting the use of germ line cells is prerequisite of the development of the preimplantation/preconception genetic CF diagnosis. A number of safety and efficacy issues have to be addressed for all approaches before human trials can be implemented.
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PMID:[Gene therapy perspectives in cystic fibrosis]. 830 49

Chloride (Cl-) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl- secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl- channel (OR) with a conductance of 25-50 pS had been proposed to play a major role in Cl- secretion. Activation by Ca2+ and the protein kinases (PK) A and C (at less than 10 nM Ca2+) as well as inhibition by PKC (at 1 microM Ca2+) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31 +/- 4 pS (n = 25). Its open probability in 10 nM Ca2+ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at -60 mV was Ca(2+)-dependent and decreased from 29% in 1 mM Ca2+ to 2% in 10 nM Ca2+. Active OR were found in (a) 25% of patches exposed to 10 nM Ca2+, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca2+, ATP and the catalytic subunit of PKA (CAK) and (c) 67% of the patches exposed to 1 mM Ca2+, ATP plus CAK. Inhibition of voltage-activated channels by addition of PKC in 1 microM or 1 mM Ca2+ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A. 838 68

Monolayers of SV-40 immortalized human airway epithelial cell lines were stimulated with bradykinin and isoproterenol to study protein phosphorylation responses that accompany ion transport regulation in normal (BEAS) and cystic fibrosis (CF/T43) cells. Phosphorylation responses were analyzed by two-dimensional gel electrophoresis of postmicrosomal supernatant fractions of 32Pi-labeled cells. Isoproterenol increased the labeling of three phosphoproteins of M(r) 17,000, 18,000, and 37,000 that were equivalent to proteins known to undergo cAMP-dependent phosphorylation in T84 cell monolayers. Distinct proteins showed increased phosphorylation with bradykinin, including acidic proteins of M(r) 15,000 and 29,000. These resembled proteins exhibiting Ca(2+)-dependent phosphorylation T84 cells. The CF/T43 and BEAS cell protein phosphorylation responses were indistinguishable. These findings support the concept that the regulation and function of protein kinase A is normal in cystic fibrosis airway epithelia and that the abnormal cAMP-mediated regulation of chloride permeability in these cells is due to altered regulatory or effector proteins.
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PMID:Protein phosphorylation responses in normal and cystic fibrosis airway epithelial cell lines. 839 78

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.
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PMID:CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells. 857 48

Cystic fibrosis is caused by defects in a chloride-transporting protein termed cystic fibrosis transmembrane conductance regulator (CFTR). This study presents an innovative procedure to evaluate expression of functional CFTR. The technique uses the potential-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC2(3), by single-cell fluorescence imaging. The DiSBAC2(3) method was first validated on the mouse mammary tumor cell line C127, stably expressing wild-type CFTR. Activation of protein kinase A by the cAMP-permeable analogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR. The DiSBAC2(3) method is quick, simple, and reproducible, and does not require invasive cell loading procedures. The system was then applied to the cell model of the human lung tumor cell line A549, in which exogenous CFTR was expressed by infecting with the replication-deficient recombinant adenovirus AdCFTR. DiSBAC2(3) was able to detect the fraction of cells in which the expression of CFTR protein was confirmed by immunocytochemistry. The DiSBAC2(3) probe was also used in human nasal respiratory cells cultured in vitro, in which it efficiently discriminated between endogenous CFTR in normal and CF cells. Functional evaluation of CFTR function by the described method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.
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PMID:Use of a membrane potential-sensitive probe to assess biological expression of the cystic fibrosis transmembrane conductance regulator. 859 Jul 31

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.
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PMID:Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP. 859 59

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.
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PMID:Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells. 866 2

Patch-clamp, iodide efflux, and biochemical techniques were used to evaluate the ability of phenylimidazothiazoles to open normal and mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels and to investigate the mechanism of activation. As reported previously for bromotetramisole, levamisole activated wild-type CFTR channels stably expressed in Chinese hamster ovary cells in the absence of other secretagogues and without elevating intracellular cAMP or calcium. The protein kinase A (PKA) inhibitor N - (2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesul-fonamid e abolished activation by forskolin but only partially inhibited stimulation by levamisole, suggesting the involvement of other kinases. CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DeltaF508) all responded to phenylimidazothiazoles. Moreover, levamisole and bromotetramisole increased the activity of wild-type and mutant channels already exposed to PKA + MgATP, consistent with the inhibition of a constitutive, membrane-associated phosphatase activity. We conclude that phenylimidazothiazole drugs can open normal and mutated CFTR channels by stabilization of phosphoforms of CFTR that are produced by basal activity of PKA and alternative protein kinases. If similar stimulation is observed in humans in vivo, phenylimidazothiazoles may be useful in the development of pharmacological therapies for cystic fibrosis.
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PMID:cAMP- and Ca2+-independent activation of cystic fibrosis transmembrane conductance regulator channels by phenylimidazothiazole drugs. 866 98

The anion-selective channel CFTR (cystic fibrosis transmembrane conductance regulator), whose dysfunction is responsible for the onset of cystic fibrosis, is regulated by cAMP through the activation of protein kinase A (PKA). The nature of this activation process is unknown. In the present study, patch-clamp techniques were applied to both mouse mammary adenocarcinoma cells expressing human epithelial CFTR (CFTR cells) and cultured neonatal rat ventricular myocytes (NRVM), to determine whether CFTR is modulated by the actin cytoskeleton, and whether the actin cytoskeleton may be implicated in the cAMP-stimulated activation of the channel protein. Acute changes in the actin cytoskeleton by addition of cytochalasin D (CD) activated whole-cell currents in CFTR cells and NRVM. Addition of actin to excised, inside-out patches also activated CFTR. A functional characterization of CFTR in either cell type included cAMP-induced, linear whole-cell and single-channel currents in symmetrical Cl-, permeability to ATP, and inhibition by either diphenylamine-carboxylate (DPC) or a monoclonal antibody raised against CFTR. Incubation of CFTR cells and NRVM with CD for over 6 h prevented CFTR activation either by the cAMP pathway under whole-cell conditions or by PKA under excised inside-out conditions. Thus a complete derangement of the actin cytoskeleton prevents the cAMP-dependent activation of CFTR. CFTR activation, however, was restored by subsequent addition of actin. In summary, changes in actin filament organization modulate CFTR channel activity by a mechanism entailing a direct interaction between actin filaments and CFTR.
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PMID:Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator. 873 83

Using an 125I- efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca(2+)-sensitive Cl- conductance, triggered upon stimulation of the cells with endothelin-1 or Ca(2+)-ionophore; (2) a cAMP/protein kinase A-operated Cl- conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI- channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl- channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl- channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.
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PMID:Expression and regulation of chloride channels in neonatal rat cardiomyocytes. 873 39


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