B56 delta subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production

Abstract

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser(1179) phosphorylation, aphidicolin decreased eNOS-Ser(116) phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56 delta of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser(116) dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56 delta-Ser(566) phosphorylation, although expression of neither the catalytic subunit C alpha (PP2A C alpha) nor B56 delta was altered. Ectopic expression of dominant negative (dn)-B56 delta reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser(116) and B56 delta-Ser(566). Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56 delta. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser(116), at least in part by activating PP2A B56 delta, resulting in NO release in BAEC. (C) 2015 Elsevier Inc. All rights reserved.