Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes
Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes
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DOI：
10.1242/jcs.00889
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发表时间：
2004-01-26
影响因子：
4
通讯作者：
Kardami, E
中科院分区：
文献类型：
--
作者：
Doble, BW;Dang, XT;Kardami, E
心肌细胞的促有丝分裂刺激与缝隙连接偶联和蛋白激酶C（PKC）介导的差距连接蛋白连接蛋白43（Cx43）磷酸化的降低有关。识别和干扰响应刺激而磷酸化的氨基酸是确定Cx43磷酸化和细胞周期之间关系的重要步骤。使用免疫印迹和磷酸特异性抗体，我们能够表明，Cx43上的丝氨酸-262（S262）在心肌细胞的生长因子或PKC刺激下变得磷酸化。为了检测Cx43、S262磷酸化和细胞-细胞接触（和/或偶联）对DNA合成的影响，我们在细胞-细胞接触形成的培养物或分离的心肌细胞中过表达野生型（wt）或突变型Cx43，其携带S262-丙氨酸（S262 A，模拟未磷酸化状态）或S262-天冬氨酸（S262 D，模拟组成性磷酸化）取代。wt-Cx43的过表达引起DNA合成的显着减少，而不管细胞-细胞接触的存在。在细胞-细胞接触形成培养物中，S262 D突变逆转，而S262 A突变增加Cx43的抑制作用。在没有细胞-细胞接触的情况下，S262-Cx43突变对Cx43抑制DNA合成没有显著影响。通过刮片加载评价的染料偶联表明S262 A过表达的肌细胞中间隙连接通透性增加（与野生型或S262 D相比）。我们的结论是，Cx43抑制心肌细胞DNA的合成，无论细胞-细胞接触或耦合。然而，为了逆转S262磷酸化对Cx43 DNA合成的抑制，需要细胞-细胞接触和可能的间隙连接介导的通讯。
Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.