Stimulated phosphorylation of intracellular connexin43

A monoclonal antibody, Zymed 13-8300, was previously reported to only detect nonphosphorylated connexin43 (Nagy et al., Exp. Cell Res. 236, 127-136, 1997). We show that 13-8300 can detect several phosphorylated species of connexin43 in Western blots after stimulation of two fibroblast cell systems with fresh growth medium, 12-O-tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosphorylated connexin43 could migrate at the same position during electrophoresis. The comigration of differentially phosphorylated species may complicate the molecular and functional analysis of phosphorylation sites in Cx43. Immunofluorescence experiments indicated that the newly generated phosphorylated Cx43 forms mainly had a perinuclear location. Also, in cells treated with brefeldin A for 8 h, in which the majority of connexin43 was intracellular, phosphorylation was induced by the agents. Phosphorylation of intracellular connexin43 can therefore be induced by several stimuli.