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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.
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PMID:Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line. 133 9

1. Recent discoveries have implicated regulation of an apical membrane chloride channel as site of a defect in cystic fibrosis (CF). The channel fails to respond to stimuli that elevate intracellular cAMP. 2. This paper describes properties of reversible cycles of protein phosphorylation and considers substrate specificity, reactions with model peptides, and space-filling structural models. 3. Mutation of a channel regulatory protein is proposed to involve either: (a) change of phosphorylated serine residue to an unreactive residue, (b) change in a nearby residue that does not affect phosphorylation by cAMP-dependent kinase, but results in dephosphorylation by a different phosphatase, or (c) change in a nearby residue that produces a structure unreactive with cAMP-dependent protein kinase. 4. Perhaps in CF sidechains with branched structures at the beta carbons occur on either side of the phosphorylated serine, like in glycogen phosphorylase, and prohibit reaction of a regulatory protein with cAMP-dependent protein kinase.
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PMID:Molecular defects in ion channel regulation in cystic fibrosis predicted from analysis of protein phosphorylation/dephosphorylation. 245 79

Abnormalities of epithelial function in cystic fibrosis (CF) have been linked to defects in cell membrane permeability to chloride or sodium ions. Recently, a class of chloride channels in airway epithelial cells have been reported to lack their usual sensitivity to phosphorylation via cAMP-dependent protein kinase, suggesting that CF could be due to a single genetic defect in these channels. We have examined single chloride and sodium channels in control and CF human nasal epithelia using the patch-clamp technique. The most common chloride channel was not the one previously associated with CF, but it was also abnormal in CF cells. In addition, the number of sodium channels was unusually high in CF. These findings suggest a wider disturbance of ion channel properties in CF than would be produced by a defect in a single type of channel.
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PMID:Cystic fibrosis affects chloride and sodium channels in human airway epithelia. 248 24

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

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulatory subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn(2+)-inducible manner and exhibited Zn(2+)-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSO4 markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestinal polypeptide, prostaglandin E2 and isoproterenol, whereas Zn(2+)-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation.
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PMID:Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells. 752 38

The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.
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PMID:Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein. 863 56

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
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PMID:Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells. 907 71

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, DeltaR(708-835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5'-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the DeltaR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the DeltaR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the DeltaR-CFTR channel results; Po increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.
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PMID:Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel. 934 69

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.
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PMID:A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules. 1007 49

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase- and ATP-regulated chloride channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Here we describe a mechanism that regulates CFTR channel activity, which is mediated by PDZ domains, a family of conserved protein-interaction modules. The Na(+)/H(+) exchanger regulatory factor (NHERF) binds to the cytoplasmic tail of CFTR through either of its two PDZ (PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1-2) containing the two PDZ domains increases the open probability (P(o)) of single CFTR channels in excised membrane patches from a lung submucosal gland cell line. Both PDZ domains are required for this functional effect, because peptides containing mutations in either domain are unable to increase channel P(o). The concentration dependence of the regulation by the bivalent PDZ1-2 domain is biphasic, i.e., activating at lower concentrations and inhibiting at higher concentrations. Furthermore, either PDZ domain alone or together is without effect on P(o), but either domain can competitively inhibit the PDZ1-2-mediated stimulation of CFTR. Our results support a molecular model in which bivalent NHERF PDZ domains regulate channel gating by crosslinking the C-terminal tails in a single dimeric CFTR channel, and the magnitude of this regulation is coupled to the stoichiometry of these interactions.
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PMID:Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. 1115 44


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