MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation

• Authors: Scott R. Johnstone, University of Virginia; Brett M. Kroncke, University of Virginia; Adam C. Straub, University of Virginia; Angela K. Best, University of Virginia; Clarence A. Dunn, Fred Hutchinson Cancer Research Center; Leslie A. Mitchell, Emory University; Yelena Peskova, University of Virginia; Robert K. Nakamoto, University of Virginia; Michael Koval, Emory University; Cecilia W. Lo, University of Pittsburgh; Paul D. Lampe, Fred Hutchinson Cancer Research Center; Linda Columbus, University of Virginia; Brant E. Isakson, University of Virginia
• Journal Title: FASEB Journal
• Volume: Volume 26, Number 2
• Publisher: Federation of American Society of Experimental Biology (FASEB) | 2012-04-01, Pages 201-211
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1161/CIRCRESAHA.112.272302
• Subjects: Biology, Cell
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemistry & Molecular Biology; Biology; Cell Biology; Life Sciences & Biomedicine - Other Topics

Abstract

Rationale: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. Objective: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. Methods and Results: We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE -/-) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43 CT) and produced null phosphorylation Ser>Ala (Cx43 MKA4/Cx43 CTMK4) and phospho-mimetic Ser>Asp (Cx43 MKD4/Cx43 CTMK4D) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43 ++) and Cx43 null (Cx43 -/-) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. Conclusions: We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.