Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The endogenous calmodulin-protein kinase system of sarcoplasmic reticulum terminal cisternae of rabbit fast-twitch muscle was studied. Investigation of a single Ca(2+)-channel in terminal cisternae fused to planar lipid bilayers demonstrated that the endogenous kinase inhibits the channel, although it remained unclear whether the phosphorylation sites are on the channel protein or on other junctional sarcoplasmic reticulum specific proteins [Hain et al., (1994) Biophys. J. 67, 1823-1833]. Our results, which show that two junctional sarcoplasmic reticulum specific proteins, i.e., triadin and histidine-rich Ca(2+)-binding protein, but not the ryanodine receptor/Ca(2+)-channel protein, are phosphorylated by membrane-bound 60 kDa protein kinase, seem to be able to resolve this ambiguity. Furthermore, such a probably specific protein isoform of calmodulin-protein kinase, by its substrate specificity and exposure to the cytoplasmic side of terminal cisternae at the junctional membrane domain and based on protease sensitivity, also seems to possess some of the potential requirements for a regulatory role in the functional state of the Ca(2+)-channel.
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PMID:Identification of triadin and of histidine-rich Ca(2+)-binding protein as substrates of 60 kDa calmodulin-dependent protein kinase in junctional terminal cisternae of sarcoplasmic reticulum of rabbit fast muscle. 773 13

The cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with highly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channel block by Mg2+ (-mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg2+ concentrations (-mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca2+ contraction cycle of the heart. For example, Ca2+ release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg2+. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.
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PMID:Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. 783 35

Monoclonal antibodies were used to identify and characterize a novel 90 kDa protein that was specifically localized to the junctional sarcoplasmic reticulum of rabbit skeletal muscle. Biochemical experiments show that the 90 kDa protein is an integral membrane protein of the junctional face membrane and is a substrate for the intrinsic protein kinase in triads. Immunofluorescence staining of serial transverse sections of skeletal muscle with a monoclonal antibody to the 90 kDa protein showed preferential staining of type II "fast" fibers. Specific labeling was confined to the interphase between the A- and I-bands, where the triad structure is localized. Immunoelectron microscopical labeling further indicates that the 90 kDa protein, like the ryanodine receptor/Ca(2+)-release channel and triadin, is confined to the terminal cisternae of the sarcoplasmic reticulum. Western blot analysis with a combination of monoclonal antibodies against the 90 kDa protein shows that it is specifically expressed in skeletal muscle but not in cardiac muscle or brain. Similarly, specific immunofluorescence labeling to the 90 kDa protein was not detected in ventricular myocytes or vascular smooth muscle cells. The junctional localization and phosphorylation of this protein suggest that it may play an important regulatory or structural role in the skeletal muscle triad junction.
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PMID:Characterization and ultrastructural localization of a novel 90-kDa protein unique to skeletal muscle junctional sarcoplasmic reticulum. 796 75

We have demonstrated recently that in cardiac sarcoplasmic reticulum (SR), a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates and activates the Ca(2+)-pumping ATPase (Ca(2+)-ATPase) in addition to phosphorylating the previously characterized substrates, phospholamban, and Ca2+ release channel (ryanodine receptor) (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397). The present study shows that a CaM kinase regulatory system capable of modulating SR Ca2+ pump activity through direct phosphorylation of the Ca(2+)-ATPase is functional in slow twitch but not fast twitch skeletal muscle. Incubation of SR vesicles isolated from rabbit slow twitch (soleus) and fast twitch (adductor magnus) skeletal muscles in the presence of Ca2+ and calmodulin resulted in phosphorylation of the Ca(2+)-ATPase in slow twitch muscle SR but not in fast twitch muscle SR. Exogenous CaM kinase II, which stimulated phosphorylation of the cardiac and slow twitch muscle SR Ca(2+)-ATPase, failed to phosphorylate fast twitch muscle SR Ca(2+)-ATPase. These observations demonstrate that CaM kinase-catalyzed phosphorylation of the Ca2+ pump is isoform-specific since heart and slow twitch muscle express the same Ca(2+)-ATPase isoform (SERCA2a), which is distinct from that of fast twitch muscle (SERCA1). As in the case of cardiac SR Ca(2+)-ATPase, phosphorylation of the slow twitch muscle SR Ca(2+)-ATPase (occurring at a serine residue) resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca2+ sensitivity. In addition, Ca2+/calmodulin-dependent prephosphorylation of slow twitch muscle SR resulted in a greater than 2-fold increase in its Ca2+ transport activity. In both cardiac and slow twitch muscle SR, phosphorylation of the Ca(2+)-ATPase by the endogenous CaM kinase occurred rapidly (maximum within 2 min at 37 degrees C), had similar pH optimum (8.5-9.0), temperature optimum (30 degrees C), and calmodulin concentration-dependence (k0.5 50-60 nM). cAMP-dependent protein kinase did not phosphorylate the Ca(2+)-ATPase appreciably in either cardiac or slow twitch muscle SR. These findings suggest a muscle-specific role for the membrane-associated CaM kinase in the modulation of Ca2+ uptake and release functions of the SR. In cardiac and slow twitch muscle, phosphorylation of the SR Ca(2+)-ATPase by CaM kinase might provide a novel mechanism for the modulation of the enzymatic and Ca2+ transport functions of this enzyme.
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PMID:Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. 798 62

Activation of a calmodulin (CaM)-dependent protein kinase associated with rabbit skeletal-muscle sarcoplasmic reticulum (SR) results in the phosphorylation of polypeptides of 450, 360, 165, 105, 89, 60, 34 and 20 kDa. Radioligand-binding studies indicated that a membrane-bound 60 kDa polypeptide contained both CaM- and ATP-binding domains. Under renaturing conditions on nitrocellulose blots, the 60 kDa polypeptide of the membrane exhibited CaM-dependent autophosphorylation activity, suggesting that it was the CaM-dependent protein kinase of SR. Ca2+/CaM-independent autophosphorylation of polypeptides of 62 and 45 kDa was found to occur in the light SR, whereas the Ca2+/CaM-dependent autophosphorylation activity was enriched in the heavy SR. Both these kinase activities were absent from transverse tubules, although these membranes were enriched in CaM-binding polypeptides of 160, 100 and 80 kDa. In the absence of Ca2+, CaM bound to a 33 kDa polypeptide of the membrane. The purified ryanodine receptor was not phosphorylated by the purified CaM kinase, although it was a substrate for protein kinase C. Affinity-purified antibodies to brain CaM kinase II cross-reacted with the 60 kDa polypeptide in Western blots and immunoprecipitated the 60 kDa polypeptide, along with the 360, 105, 89, 34 and 20 kDa phosphoproteins, from Nonidet-P-40-solubilized SR membranes. Antibodies raised against the 60 kDa kinase polypeptide did not cross-react with the other phosphoproteins, suggesting that these polypeptides were distinct and unrelated. Subcellular distribution of the 60 kDa kinase indicated the specific association of the polypeptide with the junctional-face membrane of SR. The CaM-dependent incorporation of 32P into various membrane proteins was inhibited by the CaM kinase II fragment (290-309), with an IC50 value of 2 nM for the inhibition of incorporation into the 60 kDa kinase polypeptide. Recent studies [Wang and Best (1992) Nature (London) 359, 739-741] have shown that a CaM kinase activity intrinsic to the membrane can inactivate the Ca(2+)-release channel of skeletal muscle SR. Since our results demonstrate that the 60 kDa polypeptide of SR is a CaM-dependent protein kinase, we suggest that this kinase, through its associations, may be responsible for gating the Ca(2+)-release channel.
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PMID:A 60 kDa polypeptide of skeletal-muscle sarcoplasmic reticulum is a calmodulin-dependent protein kinase that associates with and phosphorylates several membrane proteins. 824 Mar 1

The ryanodine receptor is the main Ca(2+)-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation of the ryanodine receptor has been proposed to be involved in the regulation of Ca2+ release. In the present study, we have examined the ability of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyclic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-dependent protein kinase (PK-G), or calmodulin-dependent protein kinase (PK-CaM). A large amount of phosphate incorporation was observed for PK-CaM (938 +/- 48 pmol of Pi/mg of purified channel protein), whereas the level of phosphorylation was considerably lower with PK-A or PK-G (345 +/- 139 and 96 +/- 6 pmol/mg respectively). In addition, endogenous PK-CaM activity co-migrates with the ryanodine receptor through several steps of purification, suggesting a strong association of the two proteins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phosphorylation was not observed in the purified ryanodine-receptor preparation. Taken together, these observations imply that PK-CaM is the physiologically relevant protein kinase, capable of phosphorylating the channel protein to a minimum stoichiometry of 2 mol of Pi per mol of tetramer.
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PMID:Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases. 825 17

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.
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PMID:Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase. 838 Mar 42

Porcine skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicle fractions enriched in the ryanodine receptor were phosphorylated in the presence of [gamma-32P]MgATP and either exogenous cAMP-dependent protein kinase (cAMP-PK), or Ca2+ plus calmodulin. Phosphorylation of the cardiac muscle ryanodine receptor in the presence of either cAMP-PK or calmodulin (6.4 and 10.6 pmol Pi/mg SR respectively) was approximately equal to or twice the [3H]ryanodine binding activity of this preparation (5.2 pmol/mg). Furthermore, cardiac muscle ryanodine receptor Pi incorporation catalyzed by cAMP-PK and calmodulin was approximately additive. In skeletal muscle SR, however, the level of cAMP-PK or calmodulin catalyzed phosphorylation of the intact ryanodine receptor (0.2 or 2.9 pmol Pi/mg SR, respectively) was much less than the [3H]ryanodine binding activity of this fraction (11.6 pmol/mg). Furthermore, Pi incorporation into the intact skeletal muscle ryanodine receptor was 3-8-fold less than that incorporated into a component of slightly lower M(r). Although this latter component comigrated with an immunoreactive fragment of the ryanodine receptor on polyacrylamide gels, it did not appear to be derived from the ryanodine receptor. We conclude that the significant phosphorylation of the cardiac muscle SR ryanodine receptor indicates a likely physiological role for protein kinase-mediated regulation of this Ca(2+)-channel. In contrast, the minimal phosphorylation of the skeletal muscle SR ryanodine receptor indicates that such a role of protein kinases is unlikely in this tissue.
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PMID:Phosphorylation of the porcine skeletal and cardiac muscle sarcoplasmic reticulum ryanodine receptor. 843 48

We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg2+ when terminal cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca2+/calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823-1833]. In this study, modulation by protein kinases and phosphatases on net Ca2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 mM MgCl2. There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incorporation of [gamma-32P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 +/- 0.1 (32P/RyR) for PKA, 0.89 +/- 0.08 for CamPK II and 0.95 +/- 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg2+] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation with protein kinases modulates calcium loading of terminal cisternae of sarcoplasmic reticulum from skeletal muscle. 852 60

1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.
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PMID:Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. 854 25


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