title: Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells.
abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 microM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170+/-32.3% of control, n=5). The ECE-1 activity (expressed as microM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 microM, 6 h) significantly increased the ECE-1 activity (0.28+/-0.02; n=3) compared to the control (0.07+/-0.02; n=3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 microM for 1 h; 0.10+/-0.01; n=3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18+/-0.01; n=3) compared to control (0.08+/-0.01; n=3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Vic. 3800, Australia. Sanjaya.Kuruppu@med.monash.edu.au
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Kuruppu
authors.forename: Natalie
authors.initials: N
authors.lastname: Tochon-Danguy
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001572:Benzofurans / Q000494:pharmacology; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D000072396:Endothelin-Converting Enzymes; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D063065:Organophosphonates / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001572:Benzofurans; C404932:CGS 35066; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D011493:Protein Kinase C; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D013755:Tetradecanoylphorbol Acetate