title: Protein kinase D activity controls endothelial nitric oxide synthesis.
abstract: Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Madrid 28040, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Aicart-Ramos
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols". CSIC-UAM, C/Arturo Duperier, Madrid 28029, Spain CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, 28029 Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Sánchez-Ruiloba
authors.affiliation: Fundación CNIC, Madrid 28029, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Gómez-Parrizas
authors.affiliation: Cardiovascular Research Unit University Francisco de Vitoria/Hospital Ramón y Cajal, Ctra Colmenar Viejo Km 9,100, Madrid 28034, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Zaragoza
authors.affiliation: Instituto de Investigaciones Biomédicas "Alberto Sols". CSIC-UAM, C/Arturo Duperier, Madrid 28029, Spain CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, 28029 Madrid, Spain tiglesias@iib.uam.es nacho@bbm1.ucm.es.
authors.forename: Teresa
authors.initials: T
authors.lastname: Iglesias
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Madrid 28040, Spain tiglesias@iib.uam.es nacho@bbm1.ucm.es.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rodríguez-Crespo
mesh_terms: D043925:Angiogenesis Inducing Agents; D000818:Animals; D019556:COS Cells; D002227:Carbazoles / Q000494:pharmacology; D002339:Carotid Arteries / Q000187:drug effects / Q000473:pathology*; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D004847:Epithelial Cells / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects*
chemical_list: D043925:Angiogenesis Inducing Agents; D002227:Carbazoles; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C081021:Go 6976; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C089212:protein kinase D; D011493:Protein Kinase C
keywords: Endothelium; Nitric Oxide; Protein kinase D; Vasodilatation; eNOS; eNOS-Ser1179 phosphorylation