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 proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.
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PMID:Functional domains of chicken gizzard myosin light chain kinase. 383 92

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

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.
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PMID:Phosphorylation of lens fiber cell membrane proteins. 388 45

Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylates serine residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 micrograms of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal insulin-stimulated S6 kinase has a Mr of approximately equal to 50,000-60,000; there is a variable amount of activity that sediments with a Mr of about 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2+ (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.05 mM), and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 micrograms/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate 13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-L1 cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of Mr 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. The data suggest that the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid, and Ca2+-independent proteolytic derivative nor the result of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.
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PMID:Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. 389 33

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.
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PMID:[Regulatory properties of phosphorylase from chicken skeletal muscle]. 407 75

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with 32P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [gamma-32P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of Mr 85,000 and 80,000 were present in varying proportions. The 32P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.
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PMID:Phosphorylation of rat liver glycogen synthase bound to the glycogen particle. 609 7

Two cAMP-independent acetyl-CoA carboxylase (ACC) protein kinases have been partially purified from rat liver cytosol and microsomal extracts. The first kinase, present in greatest activity in microsomal extracts, appears to be identical to casein kinase I by characteristic molecular size on gel filtration (Mr 40,000) and sodium dodecyl sulfate-gel electrophoresis (Mr 34,000), autophosphorylation of this single subunit, inability to efficiently utilize GTP, and resistance to inhibition by heparin and 2,3-diphosphoglycerate. The second kinase, predominant in cytosol, appears to be identical to casein kinase II by characteristic molecular size on gel filtration (Mr 150,000), an autophosphorylated subunit of Mr 25,000, a Km for GTP nearly equal to that of ATP, inhibition by heparin and 2,3 DPG, and relative substrate specificity. Despite the incorporation of up to 2 mol 32P/mol carboxylase subunit (kinase I) and 0.6 mol/subunit (kinase II), phosphorylation by either kinase causes no change in carboxylase activity. The site(s) phosphorylated by each kinase and by the cAMP-dependent protein kinase on carboxylase appear to be clustered on a Mr 16,000 cyanogen bromide peptide that is readily released on incubation with trypsin. The potential roles of these kinases in the regulation of ACC remain to be clarified.
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PMID:Phosphorylation of acetyl-coenzyme A carboxylase by casein kinase I and casein kinase II. 614 63

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.
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PMID:Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump. 614 39

The Ca2+-pumping ATPase has been isolated from calf heart sarcolemma by calmodulin affinity chromatography (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 3263-3270) as a polypeptide of Mr about 140,000. The purified enzyme has high affinity for Ca2+ in the presence of calmodulin (Km about 0.4 microM) but shifts to a low affinity state (Km about 20 microM) in its absence. Calmodulin increases also the Vmax of the enzyme. The effects of calmodulin are mimicked by phosphatidylserine and by a limited proteolytic treatment of the enzyme with trypsin. The purified ATPase can be reconstituted in asolectin liposomes, where it pumps Ca2+ with an approximate stoichiometry to ATP of 1. The purified (and reconstituted) enzyme is not phosphorylated by added ATP and cAMP-dependent protein kinase under conditions where the enzyme in situ is stimulated concomitant with the phosphorylation of the sarcolemmal membrane (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 9371-9373). Hence, the target of the regulatory phosphorylation system is not the ATPase molecule. The purified ATPase cross-reacts with an antibody raised against the erythrocyte Ca2+-pumping ATPase. Under the same conditions, the purified sarcoplasmic reticulum Ca2+-ATPase does not react. The proteolytic splitting pattern of the purified heart sarcolemma and erythrocyte enzymes are similar but not identical.
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PMID:Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma. 622 26

The functional domains of the regulatory subunit of isozyme II of cAMP-dependent protein kinase were studied. It was shown using Edman degradation that the regulatory subunit contained a phosphorylated residue which was very close in primary sequence to the site most sensitive to hydrolysis by low trypsin concentrations as postulated previously (Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M., and McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003). Catalytic subunit incorporated 0.9 mol of 32P from [gamma-32P]ATP into a preparation of regulatory subunit that contained 1.1 mol of endogenous phosphate. After phosphorylation by the catalytic subunit, the regulatory subunit contained 2.2 mol of chemical phosphate. The effects of heat denaturation upon the rate and extent of phosphorylation of the regulatory subunit were compared with the effects of these treatments upon the cAMP binding and inhibitory domains. These data suggested that the regulatory subunit required factors in addition to an intact phosphorylatable primary sequence in order for inhibitory activity to be expressed. Such factors might be part of the secondary or tertiary structure of the protein. These studies are discussed with respect to the mechanism of inhibition of catalytic activity, and a model of the regulatory subunit structure is proposed.
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PMID:Studies on functional domains of the regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase. 624 71


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