Journal of Biological Chemistry
Volume 275, Issue 47, 24 November 2000, Pages 36514-36522
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ENZYME CATALYSIS AND REGULATION
The Neuropeptide Processing Enzyme EC 3.4.24.15 Is Modulated by Protein Kinase A Phosphorylation*

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The metalloendopeptidase EC 3.4.24.15(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, GnRH1–9, 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 andk 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.

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Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.M001843200

AF314187

*

This work was supported by Molecular and Cellular Endocrinology Training Grant T32-DK07645 (to J. W. T. and M. C. L.), NIDA Training Grant 2T32-DA7135-16 (to P. M. C.), grants from the National Health and Medical Research Council of Australia (to A. I. S. and C. N. S.), National Institutes of Health Grant P30-HD28822 (to J. A. M.), Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (to E. S. F.), National Institutes of Health Grants NS37421 (to J. L. R. and M. J. G.) and NS39892 (to M. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .

Current address: Dept. of Histology and Embryology, Biomedical Science Institute, University of Sao Paulo, Sao Paulo, 05508-900, Brazil.