Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threoninescaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser 644 by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 lM). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 lM) from 10% [1.4 ± 0.24 AFU/(min 10 6 cells)] to 19% [2.54 ± 0.24 AFU/(min 10 6 cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.
Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide‐metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium‐dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187‐stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187‐stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148420: EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) and Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62161) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148437: EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) and Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62158) colocalize (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148406: Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62161) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) by pull down (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0096)
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