Spatiotemporal Analysis Of The Role That Protein Kinase A Phosphorylation Plays In Gap Junction Assembly

Gap junction (GJ) trafficking and intercellular communication are regulated through interactions of the GJ protein, connexin, with several kinases culminating in phosphorylation on specific serines or tyrosines in the connexin C‐terminus. Connexin43 (Cx43) is an important, ubiquitous and highly phosphorylated isoform present in many vertebrate organs. Activation of Protein Kinase A (PKA) causes increased trafficking of Cx43 to the plasma membrane hypothesized to occur by direct phosphorylation on S364/S365. Here, we imaged Cx43 tagged with a PKA phosphorylation FRET biosensor (Cx43‐AKAR3). A strong Cx43‐AKAR3 FRET ratio signal was found in the Golgi apparatus and not in the GJs. Phosphorylation in the Golgi increased after stimulation with forskolin and IBMX, potent stimulators of PKA activity. However, we found no overlap of the PKA FRET ratio signal with antibody labeling for phospho‐S365 before or after PKA stimulation. Inhibition of PKA with H89 decreased but does not abrogate the effect of forskolin/IBMX stimulation. However, treatment with this inhibitor after stimulation did not result in rapid dephosphorylation. Previously published work indicated that direct PKA phosphorylation of Cx43 is low, especially as compared with PKC or MAPK. Thus, PKA activation and localization was not consistent with direct S365 phosphorylation. Most likely, PKA phosphorylates another kinase that increases Cx43 trafficking to the plasma membrane and GJ assembly. Funding: GM072881 (GS), GM043154 & DK054441 (AN), GM055632 (PD) & GM103412 (NCMIR)