CHARACTERIZATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE PHOSPHORYLATION SITES ON THE CONNEXIN-43 GAP JUNCTION PROTEIN

We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increas e in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells, The kinase(s) responsible for phosph orylation and specific serine targets in connexin-43 have not been ide ntified. There are three consensus mitogen-activated protein (MAP) kin ase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitr o on tryptic peptides that comigrated with a subset of peptides from c onnexin-43 phosphorylated in vivo in cells treated with epidermal grow th factor. These data suggested that MAP kinase may phosphorylate conn exin-43 directly in vivo, We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to char acterize MAP kinase phosphorylation. Site-directed mutagenesis, phosph otryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser(255), Ser(279), and Se r(282), which correspond to the consensus sites recognized earlier. Ch aracterization of MAP kinase-mediated phosphorylation of connexin-43 h as defined potential targets for phosphorylation in vivo following act ivation of the epidermal growth factor receptor and has provided the b asis for studies of the effects of phosphorylation, at specific molecu lar sites, on the regulation of gap junctional communication.