Articles producció científica> Ciències Mèdiques Bàsiques

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

  • Identification data

    Identifier: imarina:5131806
    Authors:
    Hurtado E., Cilleros V., Just L., Simó A., Nadal L., Tomàs M., Garcia N., Lanuza M.A., Tomàs J.
    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
  • Others:

    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
    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
    Entity: Universitat Rovira i Virgili
    Journal publication year: 2017
    Publication Type: Journal Publications
  • Keywords:

    Cellular and Molecular Neuroscience,Molecular Biology,Neurosciences
    Pkc
    Phosphorylation
    Pdk1
    Neuromuscular junction
    Muscle contraction
    Cpkcβi
    Cpkcbetai
    Cpkc?i
    pkc
    pdk1
    neuromuscular junction
    muscle contraction
    cpkc?i
    Neurosciences
    Molecular biology
    Medicina ii
    Ciências biológicas ii
    Cellular and molecular neuroscience
  • Documents:

  • Cerca a google

    Search to google scholar