Laird D W, Puranam K L, Revel J P
Division of Biology, California Institute of Technology, Pasadena 91125.
Biochem J. 1991 Jan 1;273(Pt 1)(Pt 1):67-72. doi: 10.1042/bj2730067.
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 [35S]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 [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 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.
培养的心肌细胞被用于研究连接蛋白43（Cx43）的周转和翻译后修饰，Cx43是新生心肌细胞中的一种主要间隙连接蛋白。用抗Cx43抗体对[35S]甲硫氨酸标记的裂解物进行免疫沉淀，随后使用SDS/聚丙烯酰胺凝胶电泳（SDS/PAGE）和荧光自显影分析，结果显示出两条带，一条在40 kDa处，另一条在42 kDa处。用碱性磷酸酶处理[35S]甲硫氨酸标记的Cx43后，42 kDa处的条带消失，这表明它代表该蛋白的磷酸化形式。[32P]P1掺入42 kDa条带而非40 kDa条带证实了这一点。此外，还在44 kDa处鉴定出另一种对碱性磷酸酶敏感的Cx43磷酸化形式。在脉冲追踪实验中，心肌细胞中Cx43的半衰期确定为1 - 2小时。此外，发现Cx43上磷酸基团的周转速率在实验上由该蛋白的半衰期决定。磷酸基团在蛋白质整个生命周期中都能与其保持结合的这一观察结果，与在分离的成年大鼠心脏间隙连接斑块中Cx43主要被磷酸化的发现一致。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。
