Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.
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.
TenBroek
E M
EM
Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, MN 55108, USA. tenbr001@tc.umn.edu
Lampe
P D
PD
Solan
J L
JL
Reynhout
J K
JK
Johnson
R G
RG
D000818:Animals; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000648:ultrastructure; D016193:G1 Phase; D017629:Gap Junctions / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014162:Transfection
D018031:Connexin 43; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D012694:Serine