Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Department of Cell Biology and Development, Institute of Biomedical Sciences, University of São Paulo, Brazil.
FEBS Journal (Impact Factor: 4). 08/2009; 276(16):4358-71. DOI: 10.1111/j.1742-4658.2009.07144.x
Source: PubMed


Thimet oligopeptidase (EC; 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.
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Available from: Cristoforo Scavone, May 23, 2015
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    • "Briefly, HEK293 cells were plated in 35 mm plates (0.4 Â 10 6 cells/well) overnight and were transiently transfected with siRNA sequences using Lipofectamine RNAiMax Ò (Life Technologies Co., Calsbad, CA, USA). Enzymatic activity assays and western blot analysis were performed as previously described in our laboratory [15] [16] to verify the time at which there was the greatest decrease in EP24.15 expression. "
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