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)

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by >90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithio- propionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.
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PMID:Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. 937 88

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.
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PMID:Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl- currents in guinea pig ventricular myocytes. 938 36

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.
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PMID:Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry. 938 46

We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2-901/CFTR), which permits detection of cell surface expression, affected CFTR's biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2-901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2-901/CFTR required adenosine 3',5'-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2-901/ CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion and retrieval of cell membrane was unaffected by the presence of the FLAG epitope. These results indicate that this structural alteration does not affect the control mechanisms for channel gating and suggest that the fourth extracellular loop of CFTR does not contribute to the ion pore. Detection of M2-901/CFTR by a commercially available monoclonal antibody (M2), together with presentation of normal functional properties, makes M2-901/CFTR a valuable tool to evaluate CFTR protein expression and cellular location.
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PMID:FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR. 943 15

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.
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PMID:CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates. 946 68

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.
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PMID:Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. 946 38

Previous studies have shown that genistein increased cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in the presence of saturating concentrations of forskolin and calyculin A in intact cells. Possible molecular mechanisms for genistein's action include inhibition of tyrosine kinases, inhibition of serine/threonine protein phosphatases, or direct binding of genistein to CFTR. Since genistein inhibits several enzymes that hydrolyze ATP, and ATP hydrolysis is an intrinsic property of CFTR, we examined the effect of genistein on CFTR gating in excised inside-out patches from Hi-5 insect cells and NIH3T3 cells expressing recombinant CFTR. Genistein (50 microM) did not open phosphorylated CFTR channels by itself, but increased the ATP- induced CFTR channel current by approximately twofold. A similar magnitude of enhancement was observed when genistein was applied with PKI, a specific inhibitor of protein kinase A, or vanadate, a tyrosine phosphatase inhibitor, suggesting that inhibition of protein phosphatases or tyrosine kinases does not account for genistein's effects. The enhancement of channel current increased with increasing concentrations of genistein and reached a maximum at 35 microM genistein. At higher concentrations of genistein concentration, CFTR channel current decreased, resulting in a bell-shaped dose-response relationship. In the absence of genistein, both open- and closed-time histograms could be fitted with a single exponential function, yielding a mean open time (tauO) of 0.302 +/- 0.002 s, and a mean closed time (tauC) of 0.406 +/- 0.003 s. In the presence of 50 microM genistein, the open time histogram could be fitted with a double exponential function with tauO1 = 0.429 +/- 0. 003 s and tauO2 = 2.033 +/- 0.173 s. Thus, genistein induced a prolonged open state, an effect that mimics that of nonhydrolyzable ATP analogs. Closed time analysis showed that 50 microM genistein caused a prolonged closed state with a time constant of 2.410 +/- 0.035 s. We thus conclude that (a) the effects of genistein are likely caused by a direct binding of the drug to the CFTR protein, and (b) at least two binding sites are required to explain the effects of genistein: a high affinity site that decreases the closing rate and a low affinity site that reduces the opening rate.
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PMID:Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. 948 13

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel has been identified in the cardiac muscle of a number of mammalian species, including humans. The goal of this study was to begin quantifying the structural requirements necessary for arylaminobenzoate block of the CFTR channel. The cardiac cAMP-dependent Cl- current (ICl) was measured using the whole-cell arrangement of the patch-clamp technique in guinea pig ventricular myocytes during stimulation of protein kinase A with forskolin. At drug concentrations below the IC50 value for channel block, reduction of ICl by the arylaminobenzoates occurred in a strongly voltage-dependent manner with preferential inhibition of the inward currents. At higher drug concentrations, block of both the inward and outward ICl was observed. Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in drug block of the channel, with IC50 values of 47, 17, and 4 microM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)-benzoic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), respectively. Increasing the carbon chain length further with the compound 5-nitro-2-(4-phenylbutylamino)-benzoic acid, caused no additional increase in the potency of drug block (IC50 = 4 microM). Inhibition of ICl by the arylaminobenzoates was modulated by the pH of the external solution; increasing the pH from 7.4 to 10.0 greatly weakened NPPB block, whereas decreasing the pH to 6.4 enhanced block. In addition, block of ICl was observed during intracellular dialysis of NPPB, and this action was not affected by raising the external pH.
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PMID:Arylaminobenzoate block of the cardiac cyclic AMP-dependent chloride current. 949 22

1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.
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PMID:Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel. 950 2

1. Epithelial Na+ channels (ENaCs) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is activated by protein kinase A. Since cAMP-dependent activation of CFTR Cl- conductance is defective in cystic fibrosis (CF), ENaC currents are not inhibited by CFTR. This could explain the enhanced Na+ conductance found in CF. In the present study, we examined possible mechanisms of interaction between CFTR and ENaC co-expressed in Xenopus oocytes. 2. The magnitude of CFTR Cl- currents activated by 3-isobutyl-1-methylxanthine (IBMX) in oocytes co-expressing either wild-type or mutant CFTR and ENaC determined the degree of downregulation of ENaC currents. 3. The ability of CFTR to inhibit ENaC currents was significantly reduced either when extracellular Cl- was replaced by poorly conductive anions, e.g. SCN- or gluconate, or when CFTR was inhibited by diphenylamine-carboxylate (DPC, 1 mmol l-1). 4. Downregulation of ENaC was more pronounced at positive when compared with negative clamp voltages. This suggests that outward currents, i.e. influx of Cl- through activated CFTR most effectively downregulated ENaC. 5. Activation of endogenous Ca2+-activated Cl- currents by 1 micromol l-1 ionomycin did not inhibit ENaC current. This suggests that inhibition of ENaC mediated by Cl- currents may be specific to CFTR. 6. The present findings indicate that downregulation of ENaC by CFTR is correlated to the ability of CFTR to conduct Cl-. The data have implications for how epithelia switch from NaCl absorption to NaCl secretion when CFTR is activated by secretagogues.
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PMID:Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC. 951 36


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