Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells.
We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation.
Leykauf
Kerstin
K
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
Dürst
Matthias
M
Alonso
Angel
A
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D000918:Antibody Specificity; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018031:Connexin 43 / Q000276:immunology / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004234:Diuretics, Osmotic / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D008099:Liver / Q000166:cytology; D018613:Microscopy, Confocal; D009997:Osmotic Pressure; D010766:Phosphorylation / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011799:Quinazolines; D011817:Rabbits; D051381:Rats; D013012:Sorbitol / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology
D018031:Connexin 43; D004234:Diuretics, Osmotic; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011500:Protein Synthesis Inhibitors; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D013012:Sorbitol; D004815:Epidermal Growth Factor; D000841:Anisomycin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one