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)

Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.
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PMID:Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor. 131 34

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.
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PMID:Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase. 133 5

We studied beta-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [gamma-32P]ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the beta-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The beta-agonist-induced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during beta-adrenergic stimulation of cardiac myocytes.
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PMID:Phosphorylation of ryanodine receptors in rat myocytes during beta-adrenergic stimulation. 134 13

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.
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PMID:Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. 184 85

Skeletal-muscle sarcoplasmic reticulum (SR) comprises two distinct domains, corresponding to the free membrane of longitudinal SR (LSR) and the junctional membrane region of the terminal cisternae (TC), respectively. The junctional membrane contains the ryanodine receptor (RyR)/Ca(2+)-release channel and additional minor protein components that still require biochemical investigation, in relation to excitation-contraction coupling. Recent findings suggested the involvement in this process of a 170 kDa protein [Kim, Caswell, Talvenheimo & Brandt (1990) Biochemistry 29, 9281-9289], also characterized as a phosphoprotein in junctional TC in independent studies [Chu, Submilla, Inesi, Jay & Campbell (1990) Biochemistry 29, 5899-5905]. We show that this protein is a specific substrate of exogenous cyclic AMP-dependent protein kinase, that it is exposed to the outer surface of intact TC vesicles, and that it co-localizes with the RyR to the junctional membrane. Comparative analysis of LSR and TC subfractions for the 160 kDa glycoprotein sarcalumenin, using Western-blot techniques and specific monoclonal antibodies or concanavalin A as a ligand, revealed that the distribution of this protein within the SR corresponds inversely to both that of the RyR and of the 170 kDa protein. The 170 kDa protein, like sarcalumenin, stains blue with the cationic dye Stains-All and binds 45Ca2+ on blots, but it is uniquely distinguished by its ability to bind 125I-labelled low-density lipoprotein. The similarity of these properties, as well as the pI and solubility properties, to those described for the SR protein, recently purified and cloned and named histidine-rich Ca(2+)-binding protein [HCP; Hofmann, Brown, Lee, Pathak, Anderson & Goldstein (1989) J. Biol. Chem. 264, 8260-8270], makes it very likely that our protein and HCP may indeed be identical. The protein described in the present study differs from sarcalumenin because its migration in SDS/PAGE is accelerated in the presence of Ca2+, a previously reported property of other Ca(2+)-binding proteins [leMaire, Lund, Viel, Champeil & Moller (1989) J. Biol. Chem. 265, 1111-1123], arguing for Ca(2+)-induced protein-conformational changes. Kinase-dependent phosphorylation of our protein is another distinguishing feature, which, although not previously reported for HCP, is consistent with the presence of potential serine/threonine phosphorylation sites in the middle portion of the cloned HCP molecule. The finding that HCP, contrary to early views, selectively binds to the cytoplasmic side of the junctional membrane, together with its newly characterized properties, seem to provide new clues as to a possible role in electromechanical coupling and/or Ca2+ release.
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PMID:Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca(2+)- and low-density-lipoprotein-binding protein in rabbit skeletal muscle. 187 15

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.
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PMID:Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle. 216 64

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.
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PMID:Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase. 261 95

The calcium uptake and release machinery in heart SR have been characterized: (1) The calcium pump membrane is involved in energized Ca2+ uptake enabling muscle to relax. The calcium pump protein (CPP) in heart SR is modulated by protein kinase phosphorylation of phospholamban lowering the KCa2+. We conclude that in the membrane, phospholamban elevates KCa2+ of calcium pump protein. Phosphorylation of phospholamban attenuates the influence of phospholamban. In the limit, the intrinsic KCa2+ of calcium pump protein in heart and skeletal muscle are approximately the same. (2) The junctional face membrane is involved in calcium release which triggers muscle contraction. Ryanodine is a specific modulator of the Ca2+ release channels of SR which are involved in excitation-contraction coupling. The ryanodine receptor has been isolated, found to be equivalent to the feet structures, and on reconstitution into bilayers, identified as the calcium release channel of SR. The calcium release channel of SR is closed by ruthenium red and Mg2+ and opened by Ca2+ and ATP and low ryanodine concentration. The calcium release channel of SR is not effected by drugs such as nitrendipine, diltiazem and D-600 which modulate the slow inward Ca2+ channel of the plasmalemma/transverse tubule. (3) The calcium release channels from heart and skeletal muscle SR are similar but not identical (Table IV). Important differences distinguish the calcium release machinery in heart from that of skeletal muscle. 1. In heart there are two sources of calcium fluxes: a) extracellular Ca2+ enters via the plasmalemma slow inward calcium current; and b) "Ca2+ induced Ca2+ release" from SR. In skeletal muscle, SR is the single main source of calcium which enters via "Depolarization induced calcium release". 2. The calcium release channel from heart SR has a lower Mr approximately 340,000 vs 360,000 for skeletal muscle. 3. Ryanodine binding in cardiac SR is distinct from that in skeletal muscle (Fig. 7). 4. The isolated calcium release channel from heart SR is more sensitive to Ca2+ for calcium release (Hymel et al. 1988c). Significant progress has been achieved in identifying the calcium release channel of SR in heart and skeletal muscle. The focus of excitation-contraction coupling now shifts to defining the precise nature of the coupling of excitation to contraction.
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PMID:Regulation of muscle contraction and relaxation in heart. 304 48

In vertebrate skeletal muscle, excitation-contraction coupling may occur by a mechanical coupling mechanism involving protein-protein interactions between the dihydropyridine receptor (DHPR) of the transverse tubule membrane and the ryanodine receptor (RYR)/Ca2+ release channel of the sarcoplasmic reticulum membrane. We have previously shown that the cytoplasmic II-III loop peptides of the skeletal and cardiac muscle DHPR alpha 1 subunits (SDCL and CDCL, respectively) activate the skeletal muscle RYR. We now report that cyclic AMP-dependent protein kinase-mediated phosphorylation of Ser687 of SDCL yields a peptide that fails to activate the RYR, as determined in [3H]ryanodine binding and single channel measurements. The phosphorylated SDCL bound to the skeletal muscle but not cardiac muscle RYR, and the binding could be displaced by the unphosphorylated SDCL. A mutant SDCL with a Ser687-->Ala substitution failed to activate the RYR, but was still able to bind. Similarly, a Ser813-->Ala substitution in CDCL yielded a peptide that failed to activate the skeletal RYR. Use of three smaller overlapping peptides within the SDCL region identified an amino acid region from 666 to 726 including Ser687, which bound to and activated the skeletal muscle RYR. These results suggest that cyclic AMP-dependent protein kinase-mediated phosphorylation of the DHPR alpha 1 subunit may play a role in the functional interaction of the DHPR and RYR in skeletal muscle.
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PMID:Phosphorylation of dihydropyridine receptor II-III loop peptide regulates skeletal muscle calcium release channel function. Evidence for an essential role of the beta-OH group of Ser687. 762 72

In this study, we define calmodulin binding sites of skeletal, cardiac, and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR [Menegazzi, P., et al. (1994) Biochemistry 33, 9078-9084] were synthesized, and their interaction with calmodulin was monitored by fluorescent techniques. The central portions of the skeletal, cardiac, and brain RYR protomers display one high (CaM1; Kd ranging between 2.7 and 10.2 nM) and one low affinity (CaM2; Kd ranging between 116 and 142 nM) calmodulin binding site. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) was identified a few residues upstream from the putative transmembrane segment M1 or M5. Its affinity for calmodulin varied between the RYR isoforms: the cardiac RYR CaM3 displays a high affinity (9.09 +/- 1.0 nM, n = 5), while the skeletal and brain RYR CaM3 have low affinity, the lowest affinity being displayed by the brain isoform (234 +/- 39 nM, n = 3). The RYRs calmodulin binding site CaM1 encompasses the sequence Arg-His-Arg-Val(Ile)-Ser-Leu, which is phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. Phosphorylation of RYR PM1 peptides occurs on the Ser, corresponding to amino acid number 2919, 3020, and 3055 of the brain, cardiac, and skeletal RYR protomers, respectively. We found that phosphorylation of the RYR PM1 peptides was inhibited by calmodulin binding and that the formation of the PM1 peptide-calmodulin complex was inhibited by peptide phosphorylation. These data indicate that the effect of calmodulin binding to RYR CaM1 may be regulated by the phosphorylation state of the Ser residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu.
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PMID:Calmodulin binding sites of the skeletal, cardiac, and brain ryanodine receptor Ca2+ channels: modulation by the catalytic subunit of cAMP-dependent protein kinase? 771 Oct 31


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