The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation

The metalloendopeptidase EC 3.4.24.15 (EP24.15) is a neuropeptide-metaboliz ing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putati ve phosphorylation sites in the primary sequence led us to investigate whet her phosphorylation effects the specificity and/or the kinetics of substrat e cleavage. Only protein kinase A (PICA) treatment resulted in serine phosp horylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of re combinant rat EP24.15. Mutation analysis of each putative PICA site, in vit ro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were as sessed using physiological (GnRH, GnRH(1-9) bradykinin, and neurotensin) an d fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) subst rates. The most dramatic change upon PICA phosphorylation was a substrate-s pecific, 7-fold increase in both K-m and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phos phate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PICA-mediated phosphorylation. Cloning of the full-len gth mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequen ce, and conservation at serine 644, consistent with its putative functional role. Therefore, PICA phosphorylation is suggested to play a regulatory ro le in EP24.15 enzyme activity.