Stimulated phosphorylation of intracellular connexin43

A monoclonal antibody, Zymed 13-8300, was previously reported to only detec t nonphosphorylated connexin43 (Nagy et al, Exp. Cell Res. 236, 127-136, 19 97). We show that 13-8300 can detect several phosphorylated species of conn exin43 in Western blots after stimulation of two fibroblast cell systems wi th fresh growth medium, 12-O-tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosp horylated connexin43 could migrate at the same position during electrophore sis. The comigration of differentially phosphorylated species may complicat e the molecular and functional analysis of phosphorylation sites in Cx43. I mmunofluorescence experiments indicated that the newly generated phosphoryl ated Cx43 forms mainly had a perinuclear location. Also, in cells treated w ith brefeldin A for 8 h, in which the majority of connexin43 was intracellu lar, phosphorylation was induced by the agents. Phosphorylation of intracel lular connexin43 can therefore be induced by several stimuli, (C) 1999 Acad emic Press.