Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.
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PMID:Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase. 342 32

The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes an intramolecular self-phosphorylation or autophosphorylation. Autophosphorylation produces two strikingly different effects on kinase activity that are dependent on the level of ATP used in the reaction. At low but saturating levels of ATP (5 microM), autophosphorylation causes a 75% reduction in kinase activity, with the residual activity still retaining a dependence on Ca2+ and calmodulin. By contrast, at high but physiological levels of ATP (500 microM), the kinase is converted by autophosphorylation to a form that is autonomous of Ca2+ and calmodulin, with no accompanying reduction in activity. The extent of phosphate incorporation does not determine whether the kinase becomes inhibited or autonomous. Autophosphorylated kinase shows the functional change characteristic of the ATP concentration used during the reaction--inhibited at low ATP and autonomous at high ATP--even when compared at the same level of incorporated phosphate. ATP appears to regulate the site(s) phosphorylated during activation of the kinase and thereby modulates the dual effects of autophosphorylation. Events triggered by transient elevations of cellular Ca2+ may be potentiated and retained by generation of the Ca2+/calmodulin-independent protein kinase activity.
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PMID:Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. 346 20

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.
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PMID:Tubulin phosphorylation by casein kinase II is similar to that found in vivo. 347 37

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.
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PMID:Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase. 373 Mar 67

Microtubule-associated protein 2 (MAP-2) purified after microtubule assembly cycles from bovine brain had been shown to contain about 10 esterified phosphates (mol/mol), which were relatively phosphatase resistant and essentially confined to the projection domain which contributes to the visible arms on microtubules. The kinase responsible for phosphorylating these sites had not been identified. We have approached this question by using a phosphatase that releases the bulk of these residues and then determining which kinase can now add additional residues corresponding to those released. Three kinases were chosen because of their abundance in brain and/or proximity to microtubules. Of these only Ca/phospholipid-dependent kinase was able to recognize the previously occupied sites. We also found that MAP-2 isolated from rat brain without assembly cycles contained more phosphate than previously recognized, greater than 30 mol/mol, suggesting that 20 of these had been inadvertently released by phosphatase during assembly cycles. All 3 kinases (Ca/phospholipid-dependent, cAMP-dependent, and Ca/calmodulin-dependent kinase II) recognized more sites in the bovine than in the rat MAP-2.
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PMID:Calcium/phospholipid-dependent kinase recognizes sites in microtubule-associated protein 2 which are phosphorylated in living brain and are not accessible to other kinases. 394 5

The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes a self-phosphorylation or autophosphorylation reaction. Our conclusion that this reaction is autocatalytic is based on the following lines of evidence: The autophosphorylation reaction and the protein kinase activity toward other substrates are absolutely dependent on the presence of both Ca2+ and calmodulin; autophosphorylation and phosvitin kinase activity show a similar time course and indistinguishable heat lability; the reaction is a consistent property of every preparation of rat brain kinase; the reaction is present in both crude and highly purified preparations of similar kinases or isozymes from rat lung, spleen, heart, bovine brain, and a neuronal tissue from Aplysia californica, a marine mollusk; phosphorylation of the kinase subunits is not mimicked by addition of cAMP, cGMP, Ca2+ plus diglyceride, or addition of the cAMP-dependent protein kinase, and is not blocked by the heat-stable inhibitor protein of the cAMP-dependent protein kinase; and the reaction is intramolecular. Autophosphorylation results in the stoichiometric incorporation of phosphate into both the 51,000- and 60,000-dalton subunits.
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PMID:Mechanism of autophosphorylation of the multifunctional Ca2+/calmodulin-dependent protein kinase. 399 31

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.
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PMID:Protein kinase induction in Escherichia coli by bacteriophage T7. 459 95

We have found that the calcium action potentials of bag cell neurons from the abdominal ganglion of Aplysia may be enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The catalytic subunit was purified from bovine heart and shown to be effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 muM). In 11 of 16 injected cells, both the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73% and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in three different salt solutions (Na phosphate, K phosphate, or KCl). In two experiments, tetrodotoxin (50 muM) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the calcium antagonist Co(2+), however, diminished or abolished the spikes. In more than half of the experiments, the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In three experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance, or oscillations. Our data suggest that the stimulation of intracellular protein phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase enhances the excitability of bag cell neurons by modifying calcium and potassium channels or currents.
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PMID:Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture. 626 Dec 62

In an earlier study I demonstrated that rat brain cytosol contains a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates microtubule-associated protein 2 (MAP-2) but not MAP-1. Comparison of sites of phosphate incorporated in MAP-2 catalyzed by the Ca2+/calmodulin-dependent kinase activity and the cyclic AMP-dependent protein kinase activity in cytosolic extracts revealed distinct sites of phosphorylation (Schulman, H., 1984, Mol. Cell. Biol., 4:1175-1178; abstract by me and J.A. Kuret and K.H. Spitzer [1983, Fed. Proc., 42:2250]. I have now used MAP-2 as a substrate to purify the Ca2+/calmodulin-dependent protein kinase responsible for MAP-2 phosphorylation. The brain appears to contain a single predominant Ca2+/calmodulin-dependent protein kinase that phosphorylates MAP-2. The enzyme was purified to apparent homogeneity by column chromatography using DEAE-cellulose, phosphocellulose, hydroxylapatite, Sepharose 6B, and a calmodulin-Sepharose affinity column. The 580,000-dalton holoenzyme consists of 51,000- and 60,000-dalton subunits. The purified enzyme phosphorylates MAP-2 at the same "sites" that are phosphorylated in cytosolic extracts and thus has the same specificity as the activity present in cytosol. Analysis of phosphorylated MAP-2.1 and MAP-2.2, the two components of MAP-2, suggests considerable homology in their phosphorylated domains.
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PMID:Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin-dependent protein kinase. 673 24

The effects of cerebral ischemia on calcium/calmodulin-dependent kinase II (CaM kinase II) were investigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord ischemia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinase alpha and beta in the particulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cytosol and particulate fractions. CaM kinase II is a multifunctional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death. To identify a structural basis for the decrease in enzyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase alpha from particulate fractions of control and ischemic samples revealed not only reduced incorporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.
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PMID:Effect of cerebral ischemia on calcium/calmodulin-dependent protein kinase II activity and phosphorylation. 771 3


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