Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to defective Cl- conductance in epithelial cells. While the CFTR gene product has been detected in the plasma membrane, its presence and functional role in the membranes of intracellular compartments remain to be established. The purpose of the present experiments was to functionally localize CFTR in the endosomal membrane and to test the role of the associated Cl- conductance in the regulation of endosomal pH (pH(en)). When using conductive protonophores, the net H+ flux across the endosomal membrane of Chinese hamster ovary (CHO) cells is limited by the movement of counterions. Thus, ionic permeability could be estimated indirectly, from the changes in pH(en) determined fluorimetrically. Measurements in situ and in a cell-free microsomal preparation indicate the presence of a protein kinase A (PKA)-activated anion conductance in endosomes from CHO cells transfected with CFTR, but not in endosomes from wild-type or mock-transfected cells. In endosomes isolated from CFTR-expressing cells, the stimulatory effect of PKA was diminished by a specific peptide inhibitor of PKA, by alkaline phosphatase treatment or by a monoclonal antibody against the second nucleotide binding fold of CFTR. Increasing counterion permeability by phosphorylation of CFTR or by addition of valinomycin failed to alter the rate or extent of endosomal acidification in situ. Our observations indicate that functional CFTR, susceptible to activation by PKA, is present in endosomes of transfected CHO cells. More importantly, the data suggest that factors other than counterion permeability are the major determinants of pH(en).
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PMID:The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH. 137 35

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

AlgR is a transcriptional regulator of mucoidy in Pseudomonas aeruginosa, a critical virulence factor expressed in cystic fibrosis. AlgR belongs to the superfamily of bacterial signal transduction systems, and has been shown to bind to the algD promoter, a critical point in the regulation of mucoidy. This protein, like other typical response regulators, contains highly conserved residues known to be critical for the phosphorylation and signal transduction processes. However, a typical second component interacting with AlgR has not been identified. Here we demonstrate that AlgR undergoes phosphorylation in vitro when interacting with the well-characterized histidine protein kinase CheA. These results indicate that AlgR is capable of undergoing phosphorylation typical of other two-component signal transduction systems. Moreover, the phosphotransfer reaction between CheA and AlgR was found to be affected by the presence of carbamoyl phosphate, acetyl phosphate, and salts of phosphoramidic acid, recently shown to act as small-molecular-weight phospho-donors in the process of phosphorylation of several response regulators. These findings suggest that AlgR may react with intermediary metabolites such as carbamoyl phosphate and acetyl phosphate, and that these processes may play a role in the control of mucoidy in P. aeruginosa.
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PMID:In vitro phosphorylation of AlgR, a regulator of mucoidy in Pseudomonas aeruginosa, by a histidine protein kinase and effects of small phospho-donor molecules. 143 55

Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.
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PMID:Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia. 151 83

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.
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PMID:A cystic fibrosis pancreatic adenocarcinoma cell line. 169 30

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.
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PMID:Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals. 169 27

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by defective electrolyte transport in several epithelia. In sweat duct, pancreatic, intestinal, and airway epithelia, abnormalities in transepithelial ion transport may account for the manifestations of the disease. A Cl- impermeable apical cell membrane is a common feature in these CF epithelia. The rate of transepithelial Cl- transport is controlled in part by hormonally regulated apical membrane Cl- channels; in CF epithelia, Cl- channels are present but their regulation is defective. Most regulation studies have focused on an outwardly rectifying Cl- channel, although other channels may be involved in Cl- secretion. Phosphorylation of Cl- channels or associated regulatory proteins by cAMP-dependent protein kinase or by protein kinase C (at a low internal [Ca2+]) in excised patches of membrane activates Cl- channels in normal cells but not in CF cells. Phosphorylation with protein kinase C at a high internal [Ca2+] in excised patches of membrane inactivates the channel; such inactivation is normal in CF cells. Cl- channels can also be activated by other maneuvers including an increase in the cytosolic [Ca2+], sustained membrane depolarization, an increase in temperature, proteolysis, and changes in osmolarity; the response to such maneuvers is not defective in CF. In addition to the Cl- channel abnormalities, Na+ absorption is increased in CF epithelia. It is not certain whether the increased rate of Na+ absorption results from an increase in the number of cation channels or an alteration of their kinetics. The relation of these ion channel abnormalities to the CF gene product is unknown, but an understanding of the function of the protein product and its defective function in CF should yield important new insights into the pathogenesis and potential therapy of this disease.
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PMID:Abnormal regulation of ion channels in cystic fibrosis epithelia. 169 93

Apical membrane Cl- channels control the rate of transepithelial Cl- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl- channels in airway epithelial cells.
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PMID:Fatty acids inhibit apical membrane chloride channels in airway epithelia. 169 96

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.
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PMID:Expression and characterization of the cystic fibrosis transmembrane conductance regulator. 169 61

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.
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PMID:G-proteins mediate intestinal chloride channel activation. 170 25


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