The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C: in vivo and in vitro studies.
There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D018031:Connexin 43 / Q000276:immunology / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005347:Fibroblasts; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D015203:Reproducibility of Results
D018031:Connexin 43; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C