TURNOVER AND PHOSPHORYLATION DYNAMICS OF CONNEXIN43 GAP JUNCTION PROTEIN IN CULTURED CARDIAC MYOCYTES
TURNOVER AND PHOSPHORYLATION DYNAMICS OF CONNEXIN43 GAP JUNCTION PROTEIN IN CULTURED CARDIAC MYOCYTES
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DOI：
10.1042/bj2730067
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发表时间：
1991-01-01
影响因子：
4.1
通讯作者：
REVEL, JP
中科院分区：
文献类型：
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作者：
LAIRD, DW;PURANAM, KL;REVEL, JP
培养的心肌细胞被用来研究的周转和翻译后修饰的连接蛋白43（Cx43），在新生心肌细胞的主要间隙连接蛋白。用抗Cx43抗体免疫沉淀[S-35] Met标记的裂解物，然后用SDS/PAGE和荧光法分析，结果显示两条带，一条在40 kDa，另一条在42 kDa。[S-35] Met标记的Cx43的碱性磷酸酶处理消除了42 kDa处的条带，表明其代表蛋白质的磷酸化形式。这通过[P-32]Pi掺入42 kDa条带中而不是掺入40 kDa条带中得到证实。此外，另一种碱性磷酸酶敏感的磷酸化形式的Cx43被确定在44 kDa。在脉冲追踪实验中，Cx43在心肌细胞中的半衰期被确定为1-2小时。此外，发现Cx43上磷酸基团的周转率由蛋白质的半衰期实验定义。磷酸基团可以在蛋白质的整个生命过程中保留的观察结果与在分离的成年大鼠心脏缝隙连接斑块中发现Cx43主要被磷酸化的结果一致。我们推测，Cx43的快速周转及其多个磷酸化位点在通过间隙连接调节细胞间通讯中起重要作用。
Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [S-35]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [S-35]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [P-32]Pi incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.