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, 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.
Theproopiomelanocortin (POMC) gene and its peptide products are under complex regulation in the pituitary by multiple hormonal, neurohormonal and neurotransmitter factors. Corticotropin-releasing factor (CRF) stimulates the release of POMC-derived peptides in both anterior and intermediate lobes of the pituitary, while having differential long-term effects on levels of POMC mRNA in the two pituitary lobes in vivo. In the present study, we have analyzed the release of POMC-derived peptides, as well as changes in POMC gene transcription, primary transcript, nuclear mRNA and cytoplasmic mRNA levels following acute and chronic in vivo CRF administration in an attempt to elucidate the mechanism whereby this tissue-specific differential regulation occurs. Subcutaneous injection of CRF led to a substantial increase in plasma adrenocorticotropin, but only a minor sustained rise in plasma α-melanocyte-stimulating hormone. CRF was shown to induce rapid, time-dependent increases in POMC gene transcription in both anterior and intermediate pituitary lobes, which were reflected by increases in the level of POMC primary transcript in the nucleus. POMC primary transcript remained 2-fold elevated in the anterior lobe for at least 4 h after a single injection of CRF; in contrast, CRF stimulation of POMC primary transcript in the intermediate lobe was short-lived, and had returned to control values by 60 min after injection. After 7 days of repetitive CRF administration, POMC primary transcript, mature nuclear mRNA and cytoplasmic mRNA were 2.5 to 3.0-fold elevated in the anterior pituitary. In the intermediate lobe, long-term CRF administration did not alter the levels of POMC primary transcript, but did cause a slight (25–30%) reduction in nuclear and cytoplasmic POMC mRNA. These studies suggest that the differential regulation of POMC gene expression by CRF in anterior and intermediate pituitary lobes occurs in part due to differences in transcriptional responsiveness of the two cell types to this regulatory factor.
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