Diestel Simone, Eckert Reiner, Hülser Dieter, Traub Otto
Department of Biochemistry, Institute of Animal Anatomy and Physiology, University of Bonn, Bonn 53115, Germany.
Exp Cell Res. 2004 Apr 1;294(2):446-57. doi: 10.1016/j.yexcr.2003.11.026.
To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.
为了阐明Cx31磷酸化的作用，单独将丝氨酸残基263和266（Cx31Delta263,266）或仅266（Cx31Delta266）替换为不能被磷酸化的氨基酸。对稳定转染野生型和两种不同突变型Cx31 - cDNA构建体的HeLa细胞进行分析，检测其表达、磷酸化、定位、功能性缝隙连接通道的形成以及突变型Cx31蛋白的降解情况。与Cx31野生型相比，两种突变蛋白的磷酸化水平均显著降低，表明丝氨酸残基266对Cx31磷酸化起关键作用。这些突变均未干扰缝隙连接蛋白在细胞内的正确运输。对不同转染细胞进行脉冲追踪实验，结果显示两种突变型Cx31蛋白的周转均加快。如通过染料向相邻细胞转移以及总电导率测量（突变型Cx31Delta263,266）所示，它们的细胞间通讯减少。突变型Cx31蛋白（Cx31Delta263,266）根据其蛋白量的多少，会削弱Cx31野生型蛋白的功能，表明该突变蛋白在HeLa细胞中具有显性负效应。
