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)

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.
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PMID:Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils. 165 55

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.
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PMID:Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity. 166 9

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.
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PMID:Wortmannin, a microbial product inhibitor of myosin light chain kinase. 173 24

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.
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PMID:Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. 184 85

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.
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PMID:Mitosis-specific phosphorylation of myosin light chain kinase. 193 38

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.
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PMID:Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody. 214 59

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.
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PMID:Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver. 215 82

Cyclic GMP-binding proteins present in membrane fractions of bovine retina and, in particular, rod outer segments (ROS) were identified by photoaffinity labeling with 8-azido-[32P]cGMP. Two soluble proteins and two membrane-associated proteins were specifically labeled. The soluble proteins, 93 and 72 kDa, corresponded respectively to the alpha subunit of ROS cGMP phosphodiesterase and cGMP-dependent protein kinase. One of the two membrane-associated proteins, 53 kDa, was present in all particulate retinal fractions. Its function is unknown. It is distinct from cAMP-dependent protein kinase or the 63-kDa cGMP-activated channel from ROS. The second membrane-associated protein, 37 kDa, was present only in fractions that did not contain ROS. The molecular mass of this protein was similar to that of a cGMP-binding protein previously attributed to rod cells.
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PMID:Guanosine 3',5'-cyclic nucleotide binding proteins of bovine retina identified by photoaffinity labeling. 215 64

The major action of forskolin, the diterpine activator of adenylate cyclase, in primary (unpassaged) rat aortic smooth muscle cells is to reduce vasopressin-stimulated Ca2+ concentrations. In repetitively passaged cells, however, forskolin by itself increased Ca2+ levels by apparently stimulating Ca2+ uptake into the cell and had much smaller effects on inhibiting vasopressin-stimulated Ca2+ elevations. Both primary and passaged smooth muscle cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase was greatly reduced or absent in passaged smooth muscle cells. The introduction of purified cGMP-dependent protein kinase into the cytoplasm of passaged cells prevented forskolin from elevating intracellular Ca2+ and restored the capacity of forskolin to reduce vasopressin-stimulated Ca2+ mobilization. Similar effects were observed for isoproterenol in passaged smooth muscle cells. When introduced into cells, the active catalytic subunit of the cAMP-dependent protein kinase did not lead to reductions in Ca2+ levels. These results suggest that cAMP elevations lead to profound changes in Ca2+ metabolism through activation of both cAMP- and cGMP-dependent protein kinases. Activation of cGMP-dependent protein kinase by cAMP leads to the reduction in intracellular Ca2+, whereas activation of cAMP-dependent protein kinase may only mediate the uptake of Ca2+ from extracellular sources.
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PMID:cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells. 215 36

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.
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PMID:Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates. 215 6


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