Mitogen activated protein kinase (MAPK) phosphorylation of connexin 43 (Cx43) is associated with proliferation of vascular smooth muscle cells (VSMC) in atherosclerosis. We aimed to investigate whether MAPK phosphorylation of Cx43 directly regulates VSMC proliferation. Using in vivo models of VSMC proliferation e.g. carotid treatments with platelet-derived growth factor–BB (PDGF), we identified that MAPK phosphorylated Cx43 interacts with the cell cycle control proteins cyclin E and CDK2. To confirm this in vitro we isolated primary Cx43–/– VSMC and transfected these with constructs for Cx43 containing null phosphorylation (alanine) or phospho-mimetic (aspartate) mutations of the MAPK serines. Co-immunoprecipitation and proliferation studies in transfected cells combined with analytical size exclusion studies of purified Cx43 C-terminus and cyclin E proteins demonstrated that MAPK phosphorylation of Cx43 is critical for its binding with cyclin E and for VSMC proliferation in vitro. Finally, using a novel knock-in mouse containing Cx43 MAPK alanine mutations (Cx43-MK4A), we showed that the null-phosphorylation mutation disrupts interactions between Cx43 and cyclin E and VSMC proliferation in vivo. We conclude that MAPK phosphorylated Cx43 is a novel interacting partner of cyclin E and is required to promote VSMC proliferation
