title: The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation.
abstract: The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 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 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.
authors.affiliation: Fishberg Research Center for Neurobiology and Departments of Human Genetics and Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Tullai
authors.forename: P M
authors.initials: PM
authors.lastname: Cummins
authors.forename: A
authors.initials: A
authors.lastname: Pabon
authors.forename: J L
authors.initials: JL
authors.lastname: Roberts
authors.forename: M C
authors.initials: MC
authors.lastname: Lopingco
authors.forename: C N
authors.initials: CN
authors.lastname: Shrimpton
authors.forename: A I
authors.initials: AI
authors.lastname: Smith
authors.forename: J A
authors.initials: JA
authors.lastname: Martignetti
authors.forename: E S
authors.initials: ES
authors.lastname: Ferro
authors.forename: M J
authors.initials: MJ
authors.lastname: Glucksman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D010902:Pituitary Gland / Q000201:enzymology; D051381:Rats; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism
chemical_list: D009479:Neuropeptides; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase