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 2μM 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 μM 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 (1μM, 6h) 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; 2μM for 1h; 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.