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

• Type of Material: Article
• 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
• Language: English
• Date: 2012-04-01
• Publisher: Federation of American Society of Experimental Biology (FASEB)
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 American Heart Association, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1161/CIRCRESAHA.112.272302
• Title of Journal or Parent Work: FASEB Journal
• ISSN: 0892-6638
• Volume: 26
• Issue: 2
• Start Page: 201
• End Page: 211
• Grant/Funding Information: This work is supported by NIH HL088554 (BEI), an American Heart Association Scientist Development Grant (BEI), Phillip Morris core facilities grant (BEI), an American Heart Association Post Doctoral Award (SRJ), NRSA IF32HL103042-1 (ACS), NIH RO1GM087828 (LC), NSF MCB0845668 (LC), Jeffress Memorial Trust Research Corporation for Science Advancement for support through a Cottrell Scholar Award (LC), NIH GM55632 (PDL), NIH R01-HL083120 (MK).
• Keywords: Science & Technology; Biochemistry & Molecular Biology; Biology; Cell Biology; Life Sciences & Biomedicine - Other Topics; Life Sciences & Biomedicine
• Research Categories: Biology, Cell

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.