Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

Abstract

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extraand intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.

Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

“…Conditioned media from HUVECs (60 lL) was added to the specific substrate BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] ; (MW 1907 Da; DIIWVNTPEHVVPYGLG; Auspep, Vic, Australia), or the natural substrate BigET (MW 4283 Da) giving a final concentration of 33 or 66 lg/mL, respectively, in ECE-1 assay buffer. The reaction mixture was incubated at 37°C.…”

“…All MALDI spectra at time = 0 min confirmed the presence of BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , but neither the C-terminal cleavage product BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] (MW 1380 Da), nor the N-terminal cleavage product BigET 18-21 (MW 545 Da) were detected (data not shown). BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] was detected in the mixture containing media alone, and in the presence of thiorphan after 2 and 5 h, respectively ( Fig. 3a and b).…”

“…The reaction mixture containing BigET and conditioned media alone, as well as in the presence of inhibitors thiorphan/phosphoramidon was also analysed by MALDIToF at time = 0, 1 and 5 h. Spectra at time = 0 confirmed the presence of BigET, while endothelin-1 (ET-1) or the C-terminal cleavage product (BigET [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] ; MW 1808) was not detected (Fig. 4a).…”

“…Following reverse phase separation of the reaction mixture consisting of BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] and conditioned media, the fractions containing BigET 18-34 and BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] (m/z 1380) was analysed by tandem mass spectrometry. The amino acid sequence of the resulting peptide fragments and their corresponding molecular masses are listed in Tables 1 and 2.…”

“…Proteolytic shedding of ECE-1 was measured by monitoring the cleavage of quenched fluorescent substrate (QFS) in conditioned media. A mass spectrometry approach involving BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , was used to confirm for the first time that ECE-1, endogenously expressed by human umbilical vein endothelial cells undergoes ectodomain shedding. Our data also suggest that ADAM family of metalloproteinases or MMPs are not involved in the ECE-1 ectodomain cleavage.…”