PHOSPHORYLATION OF SERINE-2843 IN RYANODINE RECEPTOR-CALCIUM RELEASE CHANNEL OF SKELETAL-MUSCLE BY CAMP-DEPENDENT, CGMP-DEPENDENT AND CAM-DEPENDENT PROTEIN-KINASE
PHOSPHORYLATION OF SERINE-2843 IN RYANODINE RECEPTOR-CALCIUM RELEASE CHANNEL OF SKELETAL-MUSCLE BY CAMP-DEPENDENT, CGMP-DEPENDENT AND CAM-DEPENDENT PROTEIN-KINASE
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DOI：
10.1016/0167-4889(93)90023-i
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发表时间：
1993-01-17
期刊：
影响因子：
--
通讯作者：
HELLMANN, G
中科院分区：
文献类型：
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作者：
SUKO, J;MAURERFOGY, I;HELLMANN, G
本研究的目的是确定兔骨骼肌肌浆网纯化兰尼碱受体钙释放通道（RyR）被cAMP依赖性蛋白激酶（PK-A）、cGMP依赖性蛋白激酶（PK-G）和Ca2+-、CaM依赖性蛋白激酶（PK-CaM）磷酸化以及磷酸化位点的定位。 PK-A 的磷酸化程度最高（约 0.9 mol 磷酸盐/mol 受体亚基），PK-G 的磷酸化程度为二分之一到三分之二，PK-CaM 的磷酸化程度为三分之一到三分之二以上。磷酸氨基酸分析显示仅用 PK-A 和 PK-G 标记磷酸丝氨酸，用 PK-CaM 单独标记磷酸丝氨酸和磷酸苏氨酸。磷酸化 RyR 的溴化氰/胰蛋白酶消化物的反相高效液相色谱 (HPLC)（通过凝胶渗透 HPLC 纯化）和二维肽图显示了 PK-A 和 PK-G 磷酸化的一个主要磷酸肽以及 PK-CaM 磷酸化的几个标记峰。对从 PK-A 和 PK-G 磷酸化获得的主要磷酸肽以及从 PK-CaM 磷酸化获得的一种磷酸肽进行自动 Edman 序列分析，得到序列 KISQTAQTYDPR（残基 2841-2852），其中丝氨酸 2843 作为磷酸化位点（对应于共有序列 RKIS），证明所有三种蛋白激酶磷酸化中心的相同丝氨酸残基受体亚基的一个区域，建议包含钙释放通道的调节剂结合位点。
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 Ca2+-, 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.