Characterization of the Mitogen-activated Protein Kinase Phosphorylation Sites on the Connexin-43 Gap Junction Protein

doi:10.1074/jbc.271.7.3779

Characterization of the Mitogen-activated Protein Kinase Phosphorylation Sites on the Connexin-43 Gap Junction Protein (*)

From the (1)Department of Molecular Carcinogenesis, Cancer Research Center of Hawaii and the
(2)Department of Genetics and Molecular Biology, School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, the
(3)Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, and
(4)The Salk Institute, La Jolla, California 92037

^§To whom correspondence should be addressed:
Molecular Carcinogenesis, Cancer Research Center of Hawaii, 1236 Lauhala St., Honolulu, HI 96813.
Tel.: 808-586-2965; Fax: 808-586-2970; :bwcramer{at}crch.hawaii.edu.

Abstract

We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser, Ser, and Ser, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.

Footnotes

↵* This research supported by Grants CA 52098 (to A. F. L.) and CA 13884 and CA 09370 (to W. E.) from the National Cancer Institute, National Institutes of Health; Grant GM46277 from the National Institutes of Health (to Ross Johnson, University of Minnesota, supporting P. D. L.); and Grant HIFW-09-95 from the American Heart Association, Hawaii Affiliate (to B. J. W.-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GJC

gap junctional communication

Cx43

connexin-43

MAPK

mitogen-activated protein kinase

MEK

MAPK kinase

EGF

epidermal growth factor

EGFR

EGF receptor

GST

glutathione S-transferase

GST-Cx43-CT

GST fusion protein containing the carboxyl-terminal tail of Cx43

PKC

protein kinase C

PBS

phosphate buffered saline

HPLC

high performance liquid chromatography

PAGE

polyacrylamide gel electrophoresis

wt

wild type

TPCK

L-1-tosylamido-2-phenylethyl chloromethyl ketone.
↵²M. Y. Kanemitsu, L. W. M. Loo, A. F. Lau, and W. Eckhart, manuscript in preparation.
↵³P. D. Lampe, W. E. Kurata, M. Bazzi, R. Johnson, and A. F. Lau, submitted for publication.
- Received September 18, 1995.
- Revision received November 21, 1995.