Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.
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PMID:A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites. 171 1

We used the specific tyrosine kinase inhibitor genistein to define the involvement of tyrosine phosphorylation in the regulation of chloride transport in the rectal gland of the dogfish shark, a model for chloride secretion via a cystic fibrosis transmembrane conductance regulator (CFTR)-like channel. In the perfused gland, genistein (100 microM) promptly increased chloride secretion from basal values of 159 +/- 36 to 966 +/- 49 mueq.h-1.g-1 (P < 0.0001). Bumentanide fully reversed genistein-induced secretion. In primary culture monolayers of rectal gland tubular cells, genistein, but not the inactive 7-glucoside form, genistin, increased short-circuit current in a dose-dependent manner, from basal values of 2.7 +/- 4.3 to 104 +/- 10 microA/cm2 (P < 0.0001). Apically applied genistein induced significantly greater chloride secretion than basolateral addition. Genistein did not increase the adenosine 3',5'-cyclic monophosphate (cAMP) content of either perfused glands or cultured monolayers. Using an anti-phosphotyrosine antibody, we observed phosphorylation of multiple proteins. Four peptides, with molecular masses of 250, 210, 55, and 53 kDa, responded to genistein treatment with a decrease in tyrosine phosphorylation. These data demonstrate the following: 1) genistein induces bumetanide-sensitive chloride secretion in both perfused rectal glands and cultured tubular cells; 2) these effects are not accompanied by an elevation of tissue cAMP, indicating that genistein-induced secretion is not mediated by the cAMP-protein kinase A pathway; and 3) genistein-sensitive peptides are present in the rectal gland cell and are candidates for involvement in the regulation of chloride secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland. 748 46

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.
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PMID:Role of CFTR in chloride secretion across human tracheal epithelium. 749 73

We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly rectified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium. Immunoprecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regulatory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from retrovirally transduced mouse L cells using a combination of alkali stripping, Triton-X extraction, and immunoaffinity chromatography. Immunopurified CFTR proteins were reconstituted in the absence and presence of ORCC. To test the hypothesis that only functional CFTR can support activation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR505-511 peptide antibody or G551D CFTR. When anti-CFTR505-511 peptide antibodies were present prior to the addition of PKA and ATP, activation of both the ORCC and CFTR was prevented. If the antibody was added after activation of the ORCC and CFTR Cl- channels by PKA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co-incorporated into planar bilayers, only the ORCC was recorded and this channel could not be further activated by the addition of PKA and ATP. Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. We also tested the hypothesis that PKA activation of ORCC was dependent on the extracellular presence of ATP. We added ATP on the presumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inhibitory anti-CFTR505-511 antibody or G551D CFTR. In both cases the ORCC regained PKA sensitivity. Moreover, the addition of hexokinase + glucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that both the presence of CFTR as well as the presence of ATP on the extracellular side is required for activation of the ORCC by PKA and ATP.
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PMID:Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels. 749 47

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl-) transport. One possible pathway for Cl- secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl- channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl- secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (psi t) = -1.1 +/- 0.1 mV, transepithelial resistance (Rt) = 768 +/- 58 omega.cm2, and short-circuit current (Isc) = 1.9 +/- 0.2 microA/cm2 (mean +/- SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl- channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Isc, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells. 750 26

Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.
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PMID:Activation of the cystic fibrosis transmembrane conductance regulator by cGMP in the human colonic cancer cell line, Caco-2. 750 58

Inside-out apical membrane vesicles were isolated from bovine tracheal epithelium. They were enriched 13- and 18-fold in two apical membrane markers, alkaline phosphatase and gamma-glutamyltransferase, respectively, and presented a low level of contamination by basolateral and intracellular membranes. These apical membrane vesicles of homogeneous inside-out orientation were used to measure 36Cl- influx. The 36Cl- influx was found to be (i) voltage-insensitive (ii) diphenylcarboxylic acid-insensitive, and (iii) from 55 to 100% activated by cAMP-dependent protein kinase according to initial rates and accumulation capacities. This rapid and ATP-dependent activation was associated with phosphorylation of a 170-180-kDa protein but was not observed with a nonhydrolyzable nucleotide like adenosine 5'-O-(3-thiotriphosphate). Immunodetection experiments showed that the mature form of bovine cystic fibrosis transmembrane conductance regulator (CFTR) was only present in the apical membranes. As compared with the previously described characteristics of CFTR, the 36Cl- uptakes detected here are the in vitro manifestation of the functional form of bovine CFTR located at the apical level in these tracheal epithelial cells. Inside-out apical membrane vesicles, with freely accessible cytoplasmic sides and functional CFTR, offer a new model system to study CFTR.
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PMID:Functional characterization of cystic fibrosis transmembrane conductance regulator (CFTR) in apical membranes purified from bovine tracheal epithelium. 751 Feb 90

Cl- conductance of the apical membrane of airway epithelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabolism. Recent reports that cAMP-dependent Cl- conductance also requires ATP at the intracellular domains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggests that ATP concentration could mediate metabolic regulation of Cl- conductance. However, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucleotides within the cell. We have investigated the possibility that CFTR-mediated Cl- conductance is affected by the ratio of oxidized to reduced intracellular pyridine nucleotides. CFTR was expressed in airway and heterologous cells and studied under whole cell voltage clamp conditions, which permitted the intracellular NAD(P)+/NAD(P)H ratio to be varied independently of ATP concentration. In three cell types expressing CFTR, whole cell dialysis with reduced pyridine nucleotides inhibited activation of Cl- currents by forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), whereas dialysis with oxidized pyridines increased both basal and stimulated CFTR-mediated Cl- conductance. In cell-attached membrane patches, the open probability of 5-6-picosiemens Cl- channels that had been activated by forskolin and CPT-cAMP was further and reversibly increased by permeant oxidants. Neither swelling-induced whole cell K+ currents in CFTR-expressing cells nor swelling-induced whole cell Cl- currents in multidrug resistance protein-expressing cells were affected by NADPH. Pyridine nucleotide redox potential had little effect on phosphorylation of histone by protein kinase A. We conclude that CFTR Cl- conductance function can be modulated by pyridine nucleotide redox potential. This effect points to the existence of a mechanism or mechanisms by which cytosolic nucleotides other than ATP can affect plasma membrane Cl- conductance and may help explain how a passive ion conductance is linked to cellular energy metabolism.
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PMID:Pyridine nucleotide redox potential modulates cystic fibrosis transmembrane conductance regulator Cl- conductance. 751 Jun 95

For cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels to open, they must be phosphorylated by protein kinase A and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP. To test whether channel opening is linked to ATP hydrolysis, we applied VO4 and BeF3 to CFTR channels in inside-out patches excised from cardiac myocytes. These inorganic phosphate analogs interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis product, inorganic phosphate. The analogs acted only on CFTR channels opened by ATP and locked them open, increasing their mean open time by 2-3 orders of magnitude. These findings establish that opening and closing of CFTR channels are coupled to an ATP hydrolysis cycle.
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PMID:Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle. 751 48

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.
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PMID:External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism. 751 60


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