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)

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

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

Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells. This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis. It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea.
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PMID:Gates of Janus: cystic fibrosis and diarrhea. 751 20

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

Cystic fibrosis has been characterized as a defect in the regulation of cyclic AMP-dependent transepithelial chloride transport. The activation of cyclic AMP-dependent protein kinase A by cyclic AMP occurs normally in cystic fibrosis cells, but they fail to transport chloride ions in response to protein kinase A stimulation. Defective chloride secretion and abnormal electrolyte transport occurs in several organs including the lung, sweat glands, intestine and pancreas. The present work was aimed at exploring whether the same or similar regulatory systems are functional in platelets, and if they are altered or deficient in individuals with cystic fibrosis. Chloride transport in platelets from normal subjects and from cystic fibrosis patients was measured by cell sizing techniques where chloride permeability is the limiting factor. In platelets from healthy volunteers, the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid, inhibits the transport in a dose-dependent manner. The preservation of chloride transport capability is shown to be dependent upon the presence of either Ca2+ or two divalent cation substitutes, Cd2+ or Cu2+. It is also shown that in normal subjects 0.1 mumol/l prostaglandin E1, which elevates cyclic AMP 6 times and abolishes platelet aggregation, significantly enhances the rate constant of the transport. Furthermore, in five out of nine cystic fibrosis patients studied, platelet chloride transport did not respond to stimulation by prostaglandin E1.
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PMID:Cyclic AMP-related and cation-affected human platelet chloride transport regulation. 757 13

A new chloride channel has recently been identified by expression cloning (Paulmichl, M., et al. Nature 356: 238). To date there is no information available on the distribution of this channel in mammalian tissues. We cloned rat homologue of this Cl channel and found 92% identity in deduced amino-acid sequence. We studied the tissue distribution of its mRNA and regulation by protein kinases. Its mRNA was expressed in all 17 bovine tissues we studied, most abundantly in the brain. In the kidney, it was expressed more in the medulla than cortex. Dehydration up to 5 days did not change its mRNA level in the rat kidney. Both phorbol myristate acetate (PMA) and forskolin down regulated its expression in MDCK cells, suggesting that both PKC and PKA modulated its expression. The physiological role of this chloride channel remains to be clarified.
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PMID:Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinases. 768 61

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64-kDa protein, p64, identified as a component of this chloride channel was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinase A, protein kinase C, and casein kinase II. There was no significant homology to other protein (or DNA) sequences in the data base. The protein is expressed in all cells tested. Expression of its mRNA in Xenopus laevis oocytes led to the insertion of a protein with the appropriate molecular mass in microsomes but not in the plasma membrane. It is likely that p64 represents the chloride channel of intracellular organelles.
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PMID:Molecular cloning and characterization of p64, a chloride channel protein from kidney microsomes. 768 8

The past decade of research in cystic fibrosis has produced a wealth of information about the underlying defect responsible for the disease. The initial finding that the physiological disturbance in CF is one of abnormal electrolyte transport across epithelial tissues led to the elucidation of a pathway in which epithelial chloride transport is normally elicited in response to beta-adrenergic stimuli and involves the second messenger cAMP to activate protein kinase A. While that pathway was being described, work on the genetic front was concurrently providing information about the genomic location of the gene causing CF, which ultimately led to the identification and cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator. The cloned CFTR gene provided a powerful reagent to use in the next generation of cell physiology experiments, in which it was determined that CFTR is not only the substrate of PKA phosphorylation, a step previously determined to be in the activation pathway of the chloride channel, but is in fact a cAMP-dependent chloride conducting channel itself. Further analysis of the gene has shown that although there is a single mutation that accounts for most of CF, there are well over 200 other lesions within the gene that can cause disease as well. Identification of these mutations has provided information into the normal function of CFTR by studying these variants in heterologous expression systems. As a result, the molecular mechanism of CFTR function is beginning to unfold, as well as the mechanism by which particular mutations impair that function. From a clinical perspective, the research brings optimism from two directions. First, understanding how disease-causing mutations impair function may culminate in pharmacologic approaches that can restore function to some of these mutants. Second, treating the disease at the level of the gene appears to be a realistic goal: Gene transfer experiments in cultured CF cells have shown that the procedure will restore cAMP-dependent chloride conductance to the cells, laying the groundwork for somatic cell gene therapy as a feasible treatment for CF. Currently, work is rapidly progressing in developing delivery systems for this purpose. Finally, animal models that should not only aid in understanding the physiology of electrolyte transport in epithelia but should serve as indicators for tests of therapeutic approaches to treating CF are being developed, either by pharmacological means or by gene delivery protocols.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Molecular biology of cystic fibrosis. 769 8

Available data indicate that the liver is a target organ for parathyroid hormone (PTH) and that this effect is most likely mediated by PTH-induced calcium entry into hepatocytes. The present study examined the effects of both PTH-(1-84) and its amino-terminal fragment [PTH-(1-34)] on cytosolic calcium concentration ([Ca2+]i) of hepatocytes and explored the cellular pathways that mediate this potential action of PTH. Both moieties of PTH produced a dose-dependent rise in [Ca2+]i, but the effect of PTH-(1-84) was greater (P < 0.01) than an equimolar amount of PTH-(1-34). This effect required calcium in the medium and was totally [PTH-(1-34)] or partially [PTH-(1-84)] blocked by PTH antagonist ([Nle8,18,Tyr34]bPTH-(7-34)-NH2] and by verapamil or nifedipine. Sodium or chloride channel blockers did not modify this effect. 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and G protein activator also produced a dose-dependent rise in [Ca2+]i. Staurosporine abolished the effect of TPA, and both staurosporine and calphostin C partially inhibited the effect of PTH. Staurosporine and verapamil together produced greater inhibition of PTH action than each alone. Rp-cAMP, a competitive inhibitor of cAMP binding to the R subunit of protein kinase A, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, blocked the effect of both DBcAMP and PTH, but the effect of these agents was greater (P < 0.01) on DBcAMP action. G protein inhibitor and pertussis toxin partially blocked the action of PTH. The data indicate that 1) PTH increases [Ca2+]i of hepatocytes; 2) this action of the hormone is receptor mediated; 3) the predominant pathway for this PTH action is the stimulation of a G protein-adenylate cyclase-cAMP system, which then leads to stimulation of a calcium transport system inhibitable by verapamil or nifedipine or activation of L-type calcium channels; 4) activation of protein kinase C is also involved; and 5) the PTH-induced rise in [Ca2+]i is due, in major parts, to movement of extracellular calcium into the cell.
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PMID:Mechanisms of PTH-induced rise in cytosolic calcium in adult rat hepatocytes. 797 36

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.
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PMID:Hypotonicity activates a native chloride current in Xenopus oocytes. 818 3


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