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. In vitro 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.

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