Regulation of Connexin-43 Gap Junctional Intercellular Communication by Mitogen-activated Protein Kinase

doi:10.1074/jbc.273.15.9188

Regulation of Connexin-43 Gap Junctional Intercellular Communication by Mitogen-activated Protein Kinase*

From the ^‡Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, the ^‖Department of Genetics and Molecular Biology, School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, and the ^¶Department of Physiology, University of Arizona, Tucson, Arizona 85724

Abstract

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser²⁵⁵, Ser²⁷⁹, and Ser²⁸². In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser²⁵⁵, Ser²⁷⁹, and/or Ser²⁸² initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser²⁷⁹/Ser²⁸² sites is sufficient to disrupt gap junctional intercellular communication.

Footnotes

↵* This work was supported by NCI Grant CA 52098 (to A. F. L.) and NHLBI Grant HL 303470 (to J. M. B.) from the National Institutes of Health. Portions of this work were presented at the 1997 International Gap Junction Conference, Key Largo, FL, July 12–17, 1997 (1).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, 1236 Lauhala St., Honolulu, HI 96813. Tel.: 808-586-2965; Fax: 808-586-2970; E-mail:bwcramer{at}crch.hawaii.edu.
↵1 The abbreviations used are: Cx43, connexin-43; GJC, gap junctional communication; EGF, epidermal growth factor; MAP kinase, mitogen-activated protein kinase; GST, glutathioneS-transferase; MEK, MAP kinase kinase; LPA, lysophosphatidic acid; wt, wild type; K/O, knock-out; Me₂SO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; MBP, myelin basic protein; CT, carboxyl tail of Cx43 (aa 236–382); GST-Cx43-CT, GST fusion protein containing the CT of Cx43; aa, amino acid(s); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
↵2 Calero, G., Kanemitsu, M., Taffet, S. M., Lau, A. F., and Delmar, M. (1998) Circ. Res., in press.
↵3 Homma, N., Coombs, W., Taffet, S. M., Lau, A. F., and Delmar, M. (1998) Circ. Res., in press
- Received December 1, 1997.
- Revision received February 3, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.