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)

A variety of anthraquinone (anthracene-9,10-dione) derivatives inhibits rat brain Ca(2+)- and phospholipid-activated protein kinase C (PKC) of which the most potent inhibitors are mitoxantrone (1,4-dihydroxy-5,8-bis[2-(hydroxyethylamino)-ethylamino]-9,10- anthracenedione) (IC50 4 microM) and quinalizarin (1,2,5,8-tetrahydroxy-anthraquinone (IC50 4 microM). Anthraquinone derivatives with less polar substitution in positions 1 to 4 and 5 to 8 are less effective as inhibitors of PKC. Wheat germ Ca(2+)-dependent protein kinase (CDPK) assayed with a myosin light chain-based peptide substrate is much less sensitive to inhibition by anthraquinones, the most effective anthraquinone inhibitors being the 1,2,4-trihydroxy (IC50 14 microM), 1,8-dihydroxy-3-methyl (IC50 56 microM) and 1,2,5,8-tetrahydroxy (IC50 65 microM) derivatives. Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) is inhibited by a range of di-, tri- and tetrahydroxylated anthraquinones (IC50 values 2 to 53 microM), the most potent inhibitors being the more polar compounds, namely mitoxantrone (IC50 2 microM) and emodin (1,3,8-trihydroxy-6-methylanthraquinone) (IC50 8 microM). Mitoxantrone interacts with calmodulin as determined from abolition of Ca(2+)-dependent fluorescence enhancement of dansyl-calmodulin (IC50 4 microM). A range of anthraquinone derivatives inhibits the catalytic subunit of cAMP-dependent protein kinase (cAK). In a number of cases compounds acting as potent inhibitors of MLCK (such as mitoxantrone and emodin) are very poor inhibitors of cAK and vice versa.
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PMID:Inhibition of myosin light chain kinase, cAMP-dependent protein kinase, protein kinase C and of plant Ca(2+)-dependent protein kinase by anthraquinones. 146 88

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

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.
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PMID:Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells. 216 27

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.
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PMID:Selective binding of L-thyroxine by myosin light chain kinase. 290 27

Actomyosin in smooth muscle is in a quiescent state. The mechanism or mechanisms by which Ca2+ activates the actomyosin ATPase is not clear. There is sufficient evidence for the presence of enzyme systems which phosphorylate and dephosphorylate myosin light chains. The activity of the kinase that phosphorylates the myosin is regulated by cAMP-dependent protein kinase. Phosphorylated kinase has decreased affinity for calmodulin and lower activity when compared with unphosphorylated myosin light chain kinase. The activity of myosin light chain kinase is also regulated by calcium-calmodulin. In the presence of Ca2+, myosin is phosphorylated. In the absence of Ca2+, the phosphatase activity becomes dominant; the myosin remains in the unphosphorylated form under this condition. The Mg2+-ATPase of the phosphorylated myosin is activated by actin. The maximal activation of the Mg2+-ATPase by actin requires Ca2+ and tropomyosin, a protein located on the thin filament. Hence, the actin-activation of the Mg2+-ATPase requires Ca2+ even after phosphorylation by the calcium-calmodulin dependent kinase. The regulation of actin-activated ATPase activity by myosin light chain phosphorylation is depicted in the schematic diagram. Caldesmon, an actin-binding protein which also binds to calmodulin in the presence of Ca2+, has been shown to be present in thin-filaments isolated from smooth muscle. This protein inhibits actin-activated myosin ATPase activity. The release from this inhibition requires Ca2+ and calmodulin. The possibility that caldesmon is also involved in the calcium regulation of actomyosin in smooth muscle is presently under investigation in a number of laboratories.
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PMID:Regulation of actomyosin ATPase in smooth muscle. 294 44

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.
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PMID:A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver. 298 22

Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, Lys-Lys-Arg-Ala-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala ([Ala14,15]MLC(11-23] and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either D-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 microM and a Vmax of 3 X 10(-3) mumol.min-1.mg-1 compared to a Km of 12.5 microM and a Vmax of 1.43 mumol.min-1.mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 microM, comparable to 7 microM for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.
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PMID:Hydroxyamino acid specificity of smooth muscle myosin light chain kinase. 334 50

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. 379 39

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.
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PMID:Limited proteolysis of smooth muscle myosin light chain kinase. 384 33


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