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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF314187
Immunoprecipitated, 32P-labeled EP24.15 from either PC12 or AtT-20 cells, and in vitro protein kinase A (PKA) phosphorylated EP24.15 was extracted from SDS-PAGE gels [26]. 200 l of 6 M HCl was added, tubes purged with nitrogen gas, capped, and incubated at 110 °C for 1 h [32]
Whereas there are many possible mechanisms by which peptidase activity may be regulated, the presence of numerous putative phosphorylation sites on EP24.15 led us to examine whether phosphorylation plays a role in its modulation
Summary
Rat EP24.15 (accession number P24155) was expressed and purified, and site-directed mutagenesis performed using the EP24.15 expression vector, GEX-24.15 [21], as described previously [12, 22]. At least two independent preparations of wild type protein and each mutant were purified and characterized, with similar homogeneity, yields, and kinetic parameters. Samples for SDS-PAGE were heated in 2 ϫ sample buffer at 65 °C for 5 min. The proteins were separated on an 8% SDS-polyacrylamide gel as described by Laemmli [23]. Native gels were run except SDS and -mercaptoethanol were omitted from both the sample buffer and the polyacrylamide gel, and samples were not heated. The gels containing radiolabeled EP24.15 were exposed to film or to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for quantitation
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