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© The Rockefeller University Press, 0021-9525/1997//203 $5.00
The Journal of Cell Biology, Volume 137, Number 1, , 1997 203-210

Article

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization



Han-qing Xie*, Dale W. Laird{ddagger}, Tsg-Hui Chang*, and Valerie W. Hu*

* Department of Biochemistry and Molecular Biology, The George Washington University, Washington, DC, 20037; and {ddagger} Department of Anatomy and Cell Biology, McGill University, Montreal, Canada H3A-2B2

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43m. Cx43m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all 32Pi from Cx43m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

Abbreviations used in this paper: Cx43, connexin43; gap-FRAP, gap fluorescence redistribution after photobleaching (a dye coupling assay for gap junctions); GJIC, gap junction intercellular communication; HUVEC, human umbilical vein endothelial cells; PP2A, protein phosphatase-2A.

V.W. Hu is also grateful to the Glenn Foundation for Medical Research for a stipend in support of H.-q. Xie and to the American Heart Association (DC-96-GS-7) for current support of T.-H. Chang. D.W. Laird was supported by the Medical Research Council of Canada (MT 12241).

Address all correspondence to Dr. Valerie Hu, Department of Biochemistry and Molecular Biology, George Washington University Medical Center, 2300 Eye St., N.W., Washington, DC 20037. Tel.: (202) 994-8431. Fax: (202) 994-8974.

Han-qing Xie's current address is Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35295-0017.


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