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)

Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immuno-reactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that crossreacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous.
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PMID:Radioimmunoassay of bovine heart protein kinase. 5 18

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.
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PMID:Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. 164 27

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.
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PMID:Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. 184 81

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.
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PMID:Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases. 767 Nov 34

In previous studies (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397), the Ca(2+)-ATPase of cardiac muscle sarcoplasmic reticulum (SERCA2) was shown to be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase) on a serine residue, likely to be either Ser38, Ser167, or Ser531. SERCA2 and SERCA2 mutants S38A, S167A, and S531A were expressed in HEK-293 cells and tested for phosphorylation with CaM kinase. Mutant S38A was not phosphorylated, while mutants S167A and S531A were phosphorylated, suggesting that Ser38 is the site of CaM kinase phosphorylation in SERCA2. This conclusion was supported by the observation that phosphorylation of SERCA2 and mutants S167A and S531A by CaM kinase increased the Vmax for Ca2+ transport, while the Vmax for Ca2+ transport by mutant S38A was unaffected by exposure to a phosphorylation reaction mix. SERCA1, containing a potential CaM kinase phosphorylation site at Ser167 and two SERCA1 mutants, K35R plus H38S and T532S, in which potential CaM kinase sites were created, were not phosphorylated by CaM kinase, and Vmax for Ca2+ transport was unaffected by exposure to a phosphorylation reaction mix. Thus phosphorylation of Ser38 in SERCA2 results in a unique activation of Vmax for Ca2+ transport, providing a potential regulatory mechanism for Ca2+ removal from cardiac and other tissues in which SERCA2 is expressed.
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PMID:Identification of Ser38 as the site in cardiac sarcoplasmic reticulum Ca(2+)-ATPase that is phosphorylated by Ca2+/calmodulin-dependent protein kinase. 792 71

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has not been clearly established. In this study, we investigated the effects of the SR Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25-30%) by RR, and phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25-1 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, "phosphorylatable state" of the channel.
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PMID:Divergent effects of ruthenium red and ryanodine on Ca2+/calmodulin-dependent phosphorylation of the Ca2+ release channel (ryanodine receptor) in cardiac sarcoplasmic reticulum. 880 75

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

In cardiac muscle, a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca(2+)-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca(2+)-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca(2+)-ATPase has been identified as serine. Posphorylation of the Ca(2+)-ATPase is rapid and is reversible by a membrane-associated protein phosphatase, Ca(2+)-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca(2+)-sensitivity. The phosphorylation of the Ca(2+)-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA 2), but not the fast-twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca(2+)-ATPase phosphorylation by CaM kinase, ii) identification of Ser38 as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca2+ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca(2+)-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca2+ sequestration and therefore, the speed of muscle relaxation.
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PMID:Phosphorylation and regulation of the Ca(2+)-pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase. 920 41

The effect of acidosis on the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle has been investigated using phosphorylation-site-specific antibodies to this protein. Ventricular myocytes were stimulated at 0.5 Hz for 5 min, in either control (pH 7.4) or acid (pH 6.5) physiological salt solution, in the absence or presence of isoprenaline. Site-specific phosphorylation of phospholamban was determined by Western blotting. Acidosis reduced phosphorylation of Ser16 in the absence of isoprenaline, but did not alter the isoprenaline-induced phosphorylation of Ser16. In contrast, acidosis increased Thr17 phosphorylation in the absence and presence of isoprenaline. Buffering intracellular Ca2+ ([Ca2+]i) with BAPTA inhibited the increase in Thr17 phosphorylation during acidosis but had no effect on Ser16 phosphorylation. We conclude that acidosis can alter the phosphorylation of Ser16 and Thr17 by inhibition of protein kinase A, and by an acidosis-induced increase in [Ca2+]i and the subsequent activation of a Ca2+/calmodulin-dependent protein kinase, respectively. The possible effect of these changes in phosphorylation on the activity of the Ca2+-ATPase of the cardiac sarcoplasmic reticulum during acidosis is discussed.
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PMID:Acidosis alters the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle. 921 15

Despite their important role in controlling the cardiac Ca2+ homeostasis, presence and functions of individual isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the delta class in two differentiation states of the embryonic rat heart-derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the delta class (delta B, delta C, delta 4, delta 9) in adult rat heart, H9c2 myoblasts, and skeletal muscle-like H9c2 myotubes. delta C was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of delta 9 accompanied cardiac as well as skeletal muscle differentiation. Expression of delta B, however, was representative for differentiated cardiac muscle, whereas delta 4 expression coincided with differentiation into the skeletal muscle-like state. Our results demonstrate differentiation-dependent isoform expression of the delta class of the multifunctional Ca2+/calmodulin-dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the alpha 1-subunit of the L-type Ca2+ channel, the sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac delta isoforms.
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PMID:Differentiation-dependent expression of cardiac delta-CaMKII isoforms. 944 81


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