Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo N-0318, Norway.
K.G. Jebsen Centre for Cancer Immunotherapy, University of Oslo, Oslo N-0317, Norway.
Biochem J. 2018 Jan 31;475(2):455-476. doi: 10.1042/BCJ20170529.
A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. Inhibition of PKA activity resulted in decreased Cx43 phosphorylation, which was associated with reduced trophoblast fusion and differentiation. studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites that increases gap junction assembly at the plasmalemma. A combination of knockdown and reconstitution experiments and gap-fluorescence loss in photobleaching assays with mutant Cx43 containing single or double phosphoserine-mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication, trophoblast differentiation, and cell fusion.
数量有限的人类细胞可以融合形成多核合胞体。在人类胎盘的分化过程中，单核滋养细胞融合形成具有内分泌活性、非增殖性的多核合胞体。这个合胞体能覆盖胎盘，并管理母体和胎儿循环之间的营养物质和气体交换。我们最近报道蛋白激酶 A（PKA）是与埃兹蛋白和间隙连接蛋白 connexin 43（Cx43）形成的大分子信号复合物的一部分，提供 cAMP 介导的对间隙连接通讯的控制。在这里，我们检查了相关的磷酸化事件。PKA 活性的抑制导致 Cx43 磷酸化减少，这与滋养细胞融合和分化减少有关。使用肽阵列和质谱的研究，以及针对 Cx43 的丝氨酸 369 和 373 的定点突变，指出这是 PKA 的主要磷酸化位点，增加了质膜处的间隙连接组装。包含单个或双磷酸丝氨酸模拟氨基酸取代的突变 Cx43 的敲低和重建实验以及间隙荧光损失光漂白测定，证明 S369 和 S373 的磷酸化介导了间隙连接通讯、滋养细胞分化和细胞融合。
