title: Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine 1412.
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 N-terminal 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 Ser 916, 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 Ser 1412, 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.
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain; CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Sánchez-Ruiloba
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid (UCM), Madrid, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Aicart-Ramos
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain; CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: García-Guerra
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain; CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Pose-Utrilla
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid (UCM), Madrid, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rodríguez-Crespo
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain; CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Iglesias
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D061251:Primary Cell Culture; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction
chemical_list: D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C089212:protein kinase D; D011493:Protein Kinase C
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