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14‐3‐3 epsilon modulates the stimulated secretion of endopeptidase 24.15
Author(s) -
Carreño Flávia R.,
Goñi Camila N.,
Castro Leandro M.,
Ferro Emer S.
Publication year - 2005
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2004.02967.x
Subject(s) - forskolin , hek 293 cells , endopeptidase , secretion , transfection , intracellular , signal transduction , biology , microbiology and biotechnology , kinase , phosphorylation , protein kinase a , enzyme , phosphoserine , chemistry , biochemistry , cell culture , in vitro , serine , receptor , genetics
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/threonine‐scaffold 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 µ m ). Overexpression of 14‐3‐3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 µ m ) 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.