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)

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.
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PMID:The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator. 127 37

An anti-peptide antibody raised to the C-terminal sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) was used to examine CFTR immunoreactivity in the T84 colonocyte cell line. Immunoblots of T84 cell lysates detected CFTR as a 170-kDa protein that appeared as a broad band or doublet in SDS/PAGE. This protein comigrated with the predominant immunoblot signal detected in human pancreas and colon. An equivalent protein was detected as a prominent substrate for protein kinase A and for protein kinase C in T84 cell immunoprecipitates with this antibody. The immunoprecipitated protein resembled the protein detected by immunoblot in that both proteins showed the same change in electrophoretic mobility after digestion by N-Glycanase. The precipitated protein was indentified as CFTR by two criteria. First, the same protein was immunoprecipitated with an antibody to a different CFTR peptide, [Lys102]CFTR-(102-116). Second, two-dimensional phosphopeptide mapping was used to compare the immunoprecipitated protein with a bacterially expressed protein known to contain most of the predicted protein kinase A phosphorylation sites in CFTR. Because the six most prominent peptides in each map were equivalent, these maps confirm that the precipitated protein is CFTR. By using these antibodies for immunofluorescence and immunoperoxidase staining, CFTR was localized to the apical region of T84 cells grown in tumors and in monolayers. Thus, T84 cells express CFTR at sufficient levels to permit identification and immunochemical studies of this protein in its endogenously occurring form.
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PMID:Characterization of the cystic fibrosis transmembrane conductance regulator in a colonocyte cell line. 137 42

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.
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PMID:Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers. 137 3

Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells. 137 32

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.
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PMID:Phosphorylation of the cystic fibrosis transmembrane conductance regulator. 137 74

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

Epithelial cells utilize at least two types of apical Cl- channels, the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+/calmodulin-dependent Cl- channel. While phorbal ester (PMA) activates only CFTR-dependent Cl- secretion and the Ca2+ ionophore A23187 only the Ca2+/calmodulin-dependent Cl- secretion, PMA and A23187 share the ability to down-regulate expression of the CFTR gene at the transcriptional level. Since both PMA and A23187 can activate protein kinases, we hypothesized that protein kinase pathways may be involved in the regulation of CFTR gene expression. Exposure of HT-29 human colon carcinoma cells to the protein kinase C activator SC9 down-regulated CFTR mRNA levels in a dose-dependent fashion, similar to that seen with PMA. The reduction in CFTR transcript levels by SC9 and PMA was blocked by H7, an inhibitor of protein kinases. In a similar fashion, the down-regulation of CFTR transcript levels by A23187 was blocked by H7 as well as staurosporine, another protein kinase inhibitor. Interestingly, both H7 and staurosporine themselves increased CFTR mRNA levels. Quantification of CFTR gene transcription rate showed a reduction by SC9 (similar to that with PMA and A23187) that was prevented by H7 and that H7 by itself increased CFTR transcription. Together, these observations suggest that protein kinase pathways, likely including protein kinase C, are involved in the regulation of CFTR gene expression, with activation or inhibition of protein kinase activity down-regulating or up-regulating CFTR gene expression, respectively.
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PMID:Expression of the cystic fibrosis transmembrane conductance regulator gene can be regulated by protein kinase C. 137 89

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.
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PMID:Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents. 137 20

The expression of the cystic fibrosis (CF) gene on its introduction into nonepithelial somatic cells has recently been shown to result in the appearance of distinctive low conductance chloride channels stimulated by cyclic AMP (Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, S., Ackerley, C.A., Reyes, E.F., Tsui, L.-C., Rommens, J.M., Bear, C.E., and Riordan, J.R. (1991) Cell 64, 681-691; Anderson, M. P., Rich, D.P., Gregory, R.J., Smith, A.E., and Welsh, M.J. (1991) Science 251, 679-682). Since Xenopus oocytes provide a powerful system for ion channel characterization, we have examined whole cell and single channel currents in them after injection of cRNA to program the synthesis of the cystic fibrosis transmembrane conductance regulator (CFTR). This has enabled the direct demonstration that the cyclic AMP activation is mediated by protein kinase A and that CFTR is without effect on the endogenous calcium-activated chloride channels of the oocyte, which have been well characterized previously and widely used as reporters of the expression of G-protein-coupled receptors. These findings strengthen the argument that the CF gene codes for a novel regulated chloride channel rather than a regulatory protein which can modulate separate chloride channel molecules.
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PMID:Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. 171 61

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.
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PMID:Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator. 171 99


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