Xie H, Laird D W, Chang T H, Hu V W
Department of Biochemistry and Molecular Biology, The George Washington University, DC 20037, USA.
J Cell Biol. 1997 Apr 7;137(1):203-10. doi: 10.1083/jcb.137.1.203.
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.
蛋白质印迹研究表明，连接蛋白43（Cx43）是人类血管内皮细胞中主要的间隙连接蛋白之一，在有丝分裂期间会发生翻译后修饰。这种有丝分裂特异性修饰产生了一种以单一蛋白条带形式迁移的Cx43物种，被命名为Cx43（m）。Cx43（m）被证明是额外的丝氨酸/苏氨酸磷酸化的结果，证据如下：（a）碱性磷酸酶和丝氨酸/苏氨酸特异性蛋白磷酸酶-2A（PP2A）均可诱导凝胶迁移率增加；（b）PP2A可去除Cx43（m）上几乎所有的（32）P（i）。对有丝分裂细胞进行免疫荧光共聚焦显微镜观察发现，Cx43位于细胞内，而在非有丝分裂细胞中，Cx⁴³位于细胞间接触区域。染料偶联研究表明，有丝分裂的内皮细胞彼此之间以及与非有丝分裂细胞之间均未偶联。胞质分裂后，姐妹细胞恢复细胞偶联，且与从头合成蛋白质无关。Cx43的有丝分裂特异性磷酸化与间隙连接细胞间通讯的短暂丧失以及Cx43的重新分布相关，这表明一种调节间隙连接的蛋白激酶在M期具有活性。
