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:2.7.11.12 (PKG)
2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262-10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7 +/- 0.5 pS (n = 78) at room temperature. The Pcation/P(anion) ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10(-6) M and depolarization increases channel activity (NPo). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10(-4) M and 10(-3) M, ATP reduces NPo by 23% and 69%, respectively. Furthermore, since ADP (10(-3) M) reduces NPo by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a gamma-phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10(-4) M) or by cGMP-dependent protein kinase (10(-7) M) in the presence of 8-Br-cGMP (10(-5) M) and ATP (10(-4) M). The NSC channel is not sensitive to amiloride (10(-4) M cytoplasmic and/or extracellular) but flufenamic acid (10(-4) M) produces a voltage-dependent block, reducing NPo by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages. We conclude that the NCS channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.
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PMID:A calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells. 756 35

An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper. 765 13

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.
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PMID:Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. 767 14

Nuclear factor kappa B (NF-kappa B), consisting of p50 and p65, is bound to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappa B from I kappa B and the nuclear translocation of NF-kappa B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NF-kappa B activation, we have established lipopolysaccharide (LPS)-dependent NF-kappa B activation in a cell-free system using plasma membrane-enriched, cytosol, and nuclear fractions extracted from a human monocytic cell line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane-enriched fraction and cytosol was sufficient to activate NF-kappa B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappa B to the IL-8 gene kappa B site on an electrophoretic mobility shift assay. LPS-dependent NF-kappa B activation was inhibited by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase substrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II inhibitory peptides, suggesting that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s) are involved in LPS-mediated NF-kappa B activation. In addition, LPS induced the phosphorylation of I kappa B-alpha, starting at 5 min after the stimulation in a cell-free system. Moreover, the phosphorylation was inhibited by herbimycin A and tyrphostin, but not staurosporine, suggesting that these protein kinase inhibitors act at distinct steps of signal transmission. Establishment of ligand-dependent activation of NF-kappa B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.
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PMID:Establishment of lipopolysaccharide-dependent nuclear factor kappa B activation in a cell-free system. 787 68

We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.
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PMID:GTP-stimulated phosphorylation of P-glycoprotein in transporting vesicles from KB-V1 multidrug resistant cells. 791 30

Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent proteins. We demonstrated cGMP-dependent protein kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent protein kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent kinase catalytic subunit, calmodulin kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent protein kinase inhibition of HCl transport.
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PMID:Regulation of osteoclastic acid secretion by cGMP-dependent protein kinase. 798 May 15

Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was approximately 100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained > 90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase.
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PMID:Role of cAMP in the reactivation of demembranated ram spermatozoa. 802 Jan 7

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Stimulation of cGMP-dependent protein kinase I alpha by a peptide from its own sequence. An investigation by enzymology, circular dichroism and 1H NMR of the activity and structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide. 816 46

Accumulation of the weak base aminopyrine was used as an index of acid secretory activity in rat isolated parietal cells. The nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP) caused a dose-dependent inhibition of aminopyrine accumulation (half-maximally effective concentration 247 microM) which was accompanied by an increase in guanosine 3',5'-cyclic monophosphate (cGMP) but no decrease in cell viability (trypan blue), glucose oxidation or adenosine 3',5'-cyclic monophosphate (cAMP) content. Oxyhaemoglobin (37 microM), which scavenges NO, significantly reduced the inhibitory effect of SNAP (1 mM). Prior exposure of intact cells to SNAP also reduced aminopyrine accumulation in response to ATP in permeabilised cells, an effect prevented by Rp-8-bromoguanosine 3',5'-monophosphorothioate, which inhibits activation of cGMP-dependent protein kinase, but not by the Sp-isomer. NO thus inhibits secretory activity in rat parietal cells by a specific interaction that may involve cGMP.
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PMID:The nitric oxide donor, S-nitroso-N-acetyl-penicillamine, inhibits secretory activity in rat isolated parietal cells. 821 68


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