Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells.
Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.
Tesauro
M
M
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Building10/Room 6D03, Bethesda, MD 20892-1590, USA.
Thompson
W C
WC
Moss
J
J
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism*; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D019311:Staurosporine; D013997:Time Factors; D014162:Transfection
D000590:Amino Acid Chloromethyl Ketones; D002147:Calmodulin; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D047428:Protein Kinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases; D019311:Staurosporine