leupeptins and lucifer-yellow

The lens is an avascular organ in which gap junctions provide a pathway for intercellular communication, which is vital to maintain lens transparency. Connexin43 (Cx43) is the main gap-junctional protein in lens epithelial cells. Phosphorylation of connexins is implicated in the regulation of intercellular communication. The objective of this report is to determine whether phosphorylation of Cx43 in lens epithelial cells alters its resistance to degradation by a proteasome dependent mechanism.. Primary cultures of LEC were incubated with protein kinase activator (TPA) and allowed to recover either in the presence or absence of proteasome or lysosome inhibitors. The contribution of the proteasome for the degradation of the phosphorylated form of Cx43 was further investigated by metabolic labeling with 32P or [35S]-methionine. Subcellular distribution of Cx43 was evaluated by immunofluorescence using antibodies directed against Cx43. Gap junctional intercellular communication was evaluated by transfer of the dye Lucifer yellow.. Inhibitors of proteasome and lysosome both stabilize Cx43, while proteasome inhibitors preferentially stabilize the phosphorylated form of the protein. Pulse chase experiments with 32P or [35S]-methionine show that while phosphorylation destabilizes Cx43, proteasome inhibitors stabilize the phosphorylated form of the protein. Intercellular communication is inhibited by TPA and can be restored by proteasome inhibitors, probably by preventing loss of Cx43 from the plasma membrane following treatment with TPA.. Our observations support a model in which the combined action of phosphorylation and protein degradation by a proteasome dependent mechanism contribute to regulate Cx43 stability in plasma membrane and intercellular communication through gap junctions, thus adding a novel level of regulation to intercellular communication in lens epithelial cells.

Topics: Animals; Autoradiography; Blotting, Western; Cattle; Cell Communication; Cell Membrane; Cells, Cultured; Connexin 43; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Fluorescent Antibody Technique, Indirect; Gap Junctions; Half-Life; Isoquinolines; Lens, Crystalline; Leupeptins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Protein Kinases; Tetradecanoylphorbol Acetate