REGULATION OF THE CARDIAC RYANODINE RECEPTOR BY PROTEIN KINASE-DEPENDENT PHOSPHORYLATION
REGULATION OF THE CARDIAC RYANODINE RECEPTOR BY PROTEIN KINASE-DEPENDENT PHOSPHORYLATION
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DOI：
10.1093/oxfordjournals.jbchem.a123339
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发表时间：
1991-01-01
影响因子：
2.7
通讯作者：
SHIGEKAWA, M
中科院分区：
文献类型：
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作者：
TAKASAGO, T;IMAGAWA, T;SHIGEKAWA, M
外源性添加cAMP依赖性蛋白激酶（PKA）、cGMP依赖性蛋白激酶（PKG）或钙调蛋白（CaM）的催化亚基可诱导犬心脏微粒体（用1 mM [γ-P-32]ATP处理）中兰尼碱受体（Ca 2+释放通道）的快速磷酸化。加入蛋白激酶C（PKC）也磷酸化的心脏兰尼碱受体，但在一个相对缓慢的速度。 所观察到的Ryanodine受体的PKA、PKG或PKC依赖性磷酸化水平与心脏微粒体中[H-3]Ryanodine结合的最大水平相当，而CaM依赖性磷酸化水平约为4倍。 PKA、PKG和PKC的磷酸化分别使心脏微粒体中[H-3]ryanodine结合增加22+/-5，17 +/-4，15 +/-9%（平均值+/-SD，n = 4-5）。 相比之下，与单独的5 μ M CaM和5 μ M CaM加1 mM ATP孵育的微粒体分别使[H-3]ryanodine结合降低38+/-14和53+/-15%（平均+/-SD，n = 6）。 磷酸肽图谱和磷酸氨基酸分析提供的证据表明，PKA，PKG和PKC主要磷酸化丝氨酸残基在同一磷酸肽（肽1），而内源性钙调蛋白激酶磷酸化丝氨酸残基在不同的磷酸肽（肽4）。 光亲和标记的微粒体与光反应性I-125标记的钙调素显示，钙调素绑定到一个高分子量的蛋白质，这是免疫沉淀的单克隆抗体对心脏ryanodine受体。 这些结果表明，蛋白激酶依赖的磷酸化和钙调素在心肌肌浆网钙释放通道的功能中起着重要的调节作用。
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-P-32]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 [H-3]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [H-3]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-mu-M CaM alone and 5-mu-M CaM plus 1 mM ATP decreased [H-3]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 I-125-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.