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Published 24 December 2001. doi:10.1083/jcb.200102017
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© The Rockefeller University Press, 0021-9525/2001/12/1307 $5.00
The Journal of Cell Biology, Volume 155, Number 7, December 24, 2001 1307-1318

Article

 Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP

Erica M. TenBroek1, Paul D. Lampe2, Joell L. Solan2, James K. Reynhout3 and Ross G. Johnson1

1 Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, MN 55108
2 Fred Hutchinson Cancer Research Center, Seattle, WA 98109
3 Department of Biology, Bethel College, St. Paul, MN 55112

Address correspondence to Erica TenBroek, Dept. of Genetics, Cell Biology, and Development, University of Minnesota, 1445 Gortner Ave., 250 Biological Sciences, St. Paul, MN 55108. Tel.: (612) 614-7414. Fax: (612) 625-5754. E-mail: tenbr001{at}tc.umn.edu

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Key Words: connexin43; cAMP; protein kinase A; gap junction; site-directed mutagenesis


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