Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine
Publisher
Public Library of ScienceDate
2014-01-01Citation
10.1371/journal.pone.0095191
Plos One 9.4 (2014): e95191
ISSN
1932-6203DOI
10.1371/journal.pone.0095191Funded by
This work was supported by the Ministerio de Economía y Competitividad [SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R-C.]; Comunidad de Madrid [S2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas – CIBERNED, Instituto de Salud Carlos III, to T.I. Postdoctoral fellows L.S-R. and L.G-G. have been funded by research contracts from CIBERNED; Clara Aicart-Ramos is a recipient of a FPU predoctoral fellowship from Ministerio de Economía y CompetitividadProject
Comunidad de Madrid. S2010/BMD-2331/NEURODEGMODELSEditor's Version
http://dx.doi.org/10.1371/journal.pone.0095191Subjects
Neuronal Nitric Oxide Synthase; Enzyme; Neurodegeneration; Biología y Biomedicina / Biología; MedicinaRights
2014 Sánchez-Ruiloba et al.Abstract
Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (NO) production in muscles
and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an Nterminal
PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a
previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2)
constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes
autophosphorylated at Ser916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially
enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation
could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the
functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in
transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization.
However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of
these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain
was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS
interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the
activatory residue Ser1412, and that this phosphorylation increases nNOS activity and NO production in living cells. In
conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of NO, a
mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic
stroke or neurodegeneration
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Google Scholar:Sánchez-Ruiloba, Lucía
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Aicart-Ramos, Clara
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García-Guerra, Lucía
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Pose-Utrilla, Julia
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Rodríguez-Crespo, Ignacio
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Iglesias, Teresa
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