Analysis of connexin phosphorylation sites

Most connexins, the proteins that form gap junction channels, are phosphopr oteins. Connexin phosphorylation has been thought to regulate gap junctiona l protein trafficking, gap junction assembly, channel gating, and turnover. Connexin phosphorylation has been investigated in a variety of ways. Some connexins show mobility shifts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis on phosphorylation. Kinase modulators can change the level of connexin phosphorylation and affect gap junctional communication levels . Metabolic labeling of cultured cells has allowed both phosphoamino acid i dentification and generation of phosphotryptic peptide maps. However, ident ification of the location of phosphorylated residues within the connexin se quence has required either targeted peptide synthesis, in vitro phosphoryla tion of known sites, and two-dimensional comigration studies or liquid chro matographic separation and N-terminal sequencing of peptides. In addition t o these conventional methods, we discuss new applications of mass spectrome try to the identification of phosphorylated peptides and the specific resid ues phosphorylated within the connexin-derived peptide. (C) 2000 Academic P ress.