Author, as appears in the article.: Hurtado E., Cilleros V., Just L., Simó A., Nadal L., Tomàs M., Garcia N., Lanuza M.A., Tomàs J.
Department: Ciències Mèdiques Bàsiques
URV's Author/s: Cilleros Mañé, Víctor / Garcia Sancho, Maria de les Neus / Just Borràs, Laia / Lanuza Escolano, María Angel / NADAL MAGRIÑÀ, LAURA / SIMÓ OLLÉ, ANNA / Tomás Ferré, José Maria / Tomas Marginet, Marta
Keywords: Pkc Phosphorylation Pdk1 Neuromuscular junction Muscle contraction Cpkcβi Cpkcbetai Cpkc?i pkc pdk1 neuromuscular junction muscle contraction cpkc?i
Abstract: Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with µ-conotoxin GIIIB (µ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.
Thematic Areas: Neurosciences Molecular biology Medicina ii Ciências biológicas ii Cellular and molecular neuroscience
licence for use: https://creativecommons.org/licenses/by/3.0/es/
ISSN: 16625099
Author's mail: laia.just@urv.cat marta.tomas@urv.cat victor.cilleros@urv.cat josepmaria.tomas@urv.cat laia.just@urv.cat mariaangel.lanuza@urv.cat
Author identifier: 0000-0003-0473-3730 0000-0002-4151-1697 0000-0001-5690-9932 0000-0002-0406-0006 0000-0003-0473-3730 0000-0003-4795-4103
Record's date: 2024-09-07
Papper version: info:eu-repo/semantics/publishedVersion
Link to the original source: https://www.frontiersin.org/articles/10.3389/fnmol.2017.00270
Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
Papper original source: Frontiers In Molecular Neuroscience. 10 (270): 270-
APA: Hurtado E., Cilleros V., Just L., Simó A., Nadal L., Tomàs M., Garcia N., Lanuza M.A., Tomàs J. (2017). Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction. Frontiers In Molecular Neuroscience, 10(270), 270-. DOI: 10.3389/fnmol.2017.00270
Article's DOI: 10.3389/fnmol.2017.00270
Entity: Universitat Rovira i Virgili
Journal publication year: 2017
Publication Type: Journal Publications
Repositori URV