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)

Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1. The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined. The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein. The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity. Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme. CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes. CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain. CaM kinase I appeared to be encoded by a single gene. RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined. High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level. CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.
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PMID:Calcium/calmodulin-dependent protein kinase I. cDNA cloning and identification of autophosphorylation site. 825 80

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.
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PMID:Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase C and cAMP dependent protein kinase. 839 26

Tau protein is an integral component of paired helical filaments, a pathological feature of Alzheimer's disease. tau extracted from these filaments displays decreased electrophoretic mobility due to aberrant phosphorylation. Here we show that recombinant human tau can be phosphorylated by cAMP-dependent protein kinase resulting in decreased electrophoretic mobility. Phosphorylation of tau by cAMP-dependent protein kinase caused a 92% decrease in the maximum rate of tau-induced microtubule assembly. The sites of phosphorylation were identified by digesting phosphorylated tau with proteases, separating the peptides by reversed-phase HPLC, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase N-terminal sequencing. Five phosphorylation sites were identified, two of which were located within microtubule binding domains. One site was previously shown to be the sole phosphorylation site for CaM kinase II; phosphorylation at this site by CaM kinase II was sufficient to cause decreased electrophoretic mobility (Steiner, B., Mandelkow, E. M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Soling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) EMBO J. 9, 3539-3544). Thus two different second messenger-dependent protein kinases can phosphorylate tau at the same site and induce a shift in tau mobility like that seen in Alzheimer's disease.
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PMID:Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and effect on microtubule assembly. 841 21

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.
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PMID:Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. 870 79

Inhibition of type III adenylyl cyclase (III-AC) by intracellular Ca2+ in vivo provides a mechanism for attenuation of hormone-stimulated cAMP signals in olfactory epithelium, heart, and other tissues (Wayman, G. A., Impey, S., and Storm, D. R. (1995) J. Biol. Chem. 270, 21480-21486). Although the mechanism for Ca2+ inhibition of III-AC in vivo has not been defined, inhibition is not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, Ca2+ inhibition of III-AC is antagonized by KN-62, a CaM-dependent kinase inhibitor. In addition, constitutively activated CaM kinase II inhibits the enzyme. These data suggest that CaM kinase II regulates the activity of III-AC by direct phosphorylation or by an indirect mechanism involving phosphorylation of a protein that inhibits III-AC. Here we report that III-AC is phosphorylated in vivo when intracellular Ca2+ is increased and that phosphorylation is prevented by CaM-dependent kinase inhibitors. Site-directed mutagenesis of a CaM kinase II consensus site (Ser-1076 to Ala-1076) in III-AC greatly reduced Ca2+-stimulated phosphorylation and inhibition of III-AC in vivo. These data support the hypothesis that Ca2+ inhibition of III-AC is due to direct phosphorylation of the enzyme by CaM kinase II in vivo.
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PMID:Phosphorylation and inhibition of type III adenylyl cyclase by calmodulin-dependent protein kinase II in vivo. 879 67

In the adult myocardium the Ca2+ uptake and release functions of the sarcoplasmic reticulum (SR) are known to be regulated by a membrane-associated Ca2+-calmodulin-dependent protein kinase (CaM kinase) which phosphorylates the Ca2+-pumping ATPase (Ca2+ pump), Ca2+ release channel (ryanodine receptor) and the Ca2+ pump-regulatory protein, phospholamban. The role of CaM kinase during development, however, has not been examined previously. The present study investigated the ontogenetic expression of SR-associated CaM kinase in the rabbit myocardium as well as development-related changes in CaM kinase-mediated phosphorylation of the SR proteins (Ca2+ pump, Ca2+ release channel and phospholamban) involved in transmembrane Ca2+ cycling. For these experiments, cardiac muscle homogenate and SR-enriched membrane fraction derived from fetal (21- and 28-days gestation), newborn (2 days postnatal) and adult New Zealand White rabbits were used. Western immunoblotting analysis detected the presence of phospholamban, Ca2+ pump and Ca2+ release channel in homogenate and SR at all ages tested. The amount of these proteins in the SR increased substantially during fetal and postnatal development. Phosphorylation studies revealed the presence of CaM kinase-dependent phosphorylation of the Ca2+ pump, Ca2+ release channel and phospholamban as early as 21-days gestation. This phosphorylation could be elicited with the addition of only Ca2+ and calmodulin indicating the presence of a SR-associated CaM kinase as early as 21-days gestation. This was confirmed using a delta-CaM kinase II-specific antibody. Phosphorylation per unit amount of each substrate was greater in the fetus and newborn compared to adult. Phosphorylation of phospholamban could be elicited by exogenous cAMP-dependent protein kinase (PKA) at all developmental stages studied. Activation of SR CaM kinase with Ca2+ and calmodulin, or induction of phospholamban phosphorylation by exogenous PKA, resulted in stimulation of the Ca2+ uptake activity of SR in fetal, newborn and adult heart. These results demonstrate early ontogenetic expression of the Ca2+ cycling proteins and CaM kinase in the SR and the concurrent development of phosphorylation-dependent regulation of SR Ca2+ cycling.
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PMID:Ontogeny of sarcoplasmic reticulum protein phosphorylation by Ca2+--calmodulin-dependent protein kinase. 904 54

An understanding of the role of CaM kinase II in the pancreatic beta-cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta-cell in situ, and that this event may provide an important link in the mediation of Ca2+-dependent insulin secretion.
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PMID:Calcium-stimulated phosphorylation of MAP-2 in pancreatic betaTC3-cells is mediated by Ca2+/calmodulin-dependent kinase II. 934 Dec

The present study was undertaken to reveal underlying mechanisms of apoptosis in neurons using clonal neuronal cells, ML-DmBG2-c2, derived from Drosophila larval central nervous system 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, induced cell death with typical features of apoptosis such as internucleosomal DNA fragmentation, nuclear condensation and apoptotic bodies in the cells. Though H-7 is known to inhibit cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cGMP-dependent protein kinase (PKG), myosin light chain kinase (MLCK), and casein kinase I (CKI), specific inhibitors for these kinases such as H-89, calphostin C, ML-9, or CKI-7 did not induce apoptosis in the cells. Other kinases such as tyrosine kinase. PI3-kinase and Ca2+/CaM kinase II so far examined in the present study were interpreted not to be involved in the apoptotic cascade. Therefore, it is concluded that an H-7-sensitive substance(s) other than these kinases is responsible for the apoptosis in the neuronal cells. Caspase inhibitors prevented apoptosis in the cells treated with H-7. These results suggest that caspase(s) is involved downstream of the H-7-sensitive point in the cascade of the apoptosis.
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PMID:H-7-induced apoptosis in the cells of a Drosophila neuronal cell line through affecting unidentified H-7-sensitive substance(s). 970 Jul 17

Heart disease is one of the major cause of death in diabetic patients, but the pathogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to assess the significance of G protein signaling pathways in the pathogenesis of diabetic cardiomyopathy, we analyzed the expression of G proteins and the activities of second messenger dependent protein kinases: cAMP-dependent protein kinase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent protein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat heart. The expression of Galphaq was increased by slightly over 10% (P<0.05) in diabetic rat heart, while Galphas, Galphai, and Gbeta remained unchanged. The PKA activity in the heart did not change significantly but increased by 27% (P<0.01) in the liver. Insulin treatment did not restore the increased activity in the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and insulin treatment did not restore such increase. The CaM kinase II activity in the heart remained at the same level but was slightly increased in the liver (14% increase, P<0.05). These findings of increased expression of Galphaq in the streptozotocin-diabetic rat heart that are reflected by the increased level of PKC activity and insensitivity to insulin demonstrate that alteration of Galphaq may underlie, at least partly, the cardiac dysfunction that is associated with diabetes.
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PMID:Increased expression of Galphaq protein in the heart of streptozotocin-induced diabetic rats. 1063 Mar 71

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar beta-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.
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PMID:Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae. 1174 9


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