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)

We investigated the relaxant mechanisms of the cyclic AMP (cAMP)-increasing agents, isoproterenol, T-0509, forskolin and 3-isobutyl-1-methylxanthine (IBMX), on porcine coronary arteries contracted with U46619 (300 nM), a thromboxane A2 analogue, or 30 mM KCl, by measuring force simultaneously with intracellular Ca2+ concentration ([Ca2+]i) or cAMP and cyclic GMP (cGMP) levels. In U46619-contracted arteries, these agents decreased [Ca2+]i and force of contraction to almost the same extent in a concentration-dependent manner, whereas in KCl-contracted arteries these agents, except IBMX at higher concentrations, produced a relaxation with little change in [Ca2+]i. These agents all elevated tissue cAMP levels, and in addition, IBMX at higher concentrations increased cGMP levels. In Ca(2+)-free medium, these agents produced a concentration-dependent inhibition of Ca2+ release from intracellular Ca2+ stores induced by U46619 but not by 25 mM caffeine. Isoproterenol at a high concentration (3 microM) transiently decreased [Ca2+]i but steadily relaxed KCl-contracted arteries. This decrease in [Ca2+]i, but not the relaxation was inhibited by ryanodine and caffeine treatments. These results suggest that the relaxant mechanism of these agents on KCl-contracted arteries is mainly due to phosphorylation of myosin light chain kinase via cAMP-dependent protein kinase, resulting in a reduction of the Ca2+ sensitivity of contractile elements. Their relaxant mechanism in U46619-contracted arteries seems due to the inhibition of signal transduction of the agonist, resulting in a decrease in [Ca2+]i and inhibition of the Ca2+ sensitization.
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PMID:Relaxant mechanisms of cyclic AMP-increasing agents in porcine coronary artery. 751 40

Several derivatives of K-252a, a protein kinase inhibitor isolated from Nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (VSV) in BHK-21 cell cultures. Among those we tested, KT5926, which preferentially inhibits the myosin light chain kinase (MLCK), suppressed the viral replication by 95-99% at 15 microM. K-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. Other derivatives, KT5720 and KT5823, that are known to inhibit the cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, did not suppress VSV replication even at a high concentration as 15 microM. None of these inhibitors affected the Sindbis virus replication in BHK-21 cells under similar assay conditions as used for VSV. KT5926 and K-252a seemed to affect the VSV replication at the step(s) after the viral invasion, resulting in decreased viral RNA synthesis. Neither substance inhibited cellular casein kinase (CK) II which is known to be involved in phosphorylation of the nonstructural (NS) protein, a non-catalytic subunit of the viral RNA polymerase. These results suggest that the inhibition of VSV replication by KT5926 and K-252a is not a secondary effect due to generalized suppression of host cell activities, and that the VSV replication requires the KT5926-sensitive function(s) in the cell which would be performed by an enzyme(s) other than CK II.
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PMID:Studies on the antiviral activity of protein kinase inhibitors against the replication of vesicular stomatitis virus. 755 Jan 28

The motility of both intact and demembranated fowl spermatozoa was vigorous at 30 degrees C, but decreased markedly following the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), a specific inhibitor of myosin light chain kinase (MLCK). Furthermore, the presence of a MLCK substrate peptide also inhibited the motility of demembranated spermatozoa at 30 degrees C. In contrast, the addition of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (HA1004), specific inhibitors of cAMP-dependent protein kinase, did not appreciably affect the motility of either intact or demembranated spermatozoa. Cyclic AMP-dependent protein kinase substrate peptides were also ineffective for the inhibition of motility of demembranated spermatozoa at 30 degrees C. Immunoblotting of sperm extract, using an antibody to MLCK, revealed two major crossreacting proteins of 130 kDa and 61-64 kDa, which corresponded to the molecular mass of MLCK. In addition, immunogold particles, which reacted with the anti-MLCK antibody, were observed around or on the axoneme at the ultrastructural level. These results suggest that the phosphorylation of axonemal protein(s) by MLCK, or a MLCK-like protein, rather than by cAMP-dependent protein kinase, may be involved in the maintenance of fowl sperm motility at 30 degrees C.
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PMID:Regulatory mechanisms of fowl sperm motility: possible role of endogenous myosin light chain kinase-like protein. 763 95

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.
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PMID:Photoaffinity labeling of a peptide substrate to myosin light chain kinase. 773 Mar 16

MS-347a was isolated from the culture broths of Aspergillus sp. KY52178 as an inhibitor of smooth muscle myosin light chain kinase (MLCK). MS-347a inhibited the activity of chicken gizzard MLCK with an IC50 value of 9.2 microM. The inhibition was dependent on time of preincubation of MS-347a with the enzyme, suggesting irreversible inhibition. It is likely that the inhibitor binds to the catalytic domain of MLCK, since the compound inhibited not only calmodulin-dependent but also calmodulin-independent activity of MLCK. Calmodulin-dependent cyclic nucleotide phosphodiesterase, cAMP-dependent protein kinase and cGMP-dependent protein kinase were not inhibited by 150 microM MS-347a at all, although the compound inhibited protein kinase C with an IC50 value of 16 microM. MS-347b, a minor component was also isolated from the same culture broths. This minor component at 150 microM did not inhibit the activity of MLCK.
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PMID:MS-347a, a new inhibitor of myosin light chain kinase from Aspergillus sp. KY52178. 829 33

Embryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 microM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 microM H-7. However, another PKC inhibitor, staurosporine, did not inhibit the directed translocation (1 nM-1 microM). We also found evidence for the involvement of either cAMP- or cGMP-dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 microM H-9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP- or cGMP-dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML-7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 microM W-7 had no significant effect on the directed translocation, suggesting that calmodulin-dependent protein kinase is not involved. Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP-dependent protein kinase inhibitor, H-7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long lasting.
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PMID:Protein kinases are required for embryonic neural crest cell galvanotaxis. 831 67

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca2+. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0-2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca(2+)-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0-2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8-4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the Vmax of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphorylation site B (S828).
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PMID:Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase. 856 50

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.
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PMID:Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases. 887 9

The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.
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PMID:Molecular strategies for the dominant inhibition of protein kinase C. 896 21

The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identify with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.
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PMID:Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus. 904 80


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