title: Phospho-eNOS Ser-114 in human mesenchymal stem cells: constitutive phosphorylation, nuclear localization and upregulation during mitosis.
abstract: Activity of endothelial nitric oxide synthase (eNOS) is modulated by protein-protein interaction and phosphorylation at specific serine or threonine residues. Using immunofluorescence analysis we show here that proliferating mesenchymal stem cells (MSCs) derived from human bone marrow exhibit cytosolic and pronounced nuclear localization of eNOS. Examination of phosphorylated eNOS subspecies revealed that eNOS phosphorylated at Ser-114 is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser-1177 is localized at filamentous structures in the cytosol that are abundant in the perinuclear region. Phosphorylation of eNOS at Ser-114 but not at Ser-1177 was strongly increased in cells shortly before mitosis and decreased to normal level after completed cell division. Double immunofluorescence analysis revealed that subcellular localization of 8-hydroxyguanosine immunoreactivity was overlapping with eNOS phosphorylated at Ser-114 in human MSCs providing evidence that phosphorylation at this residue is linked to the generation of superoxide anions. As expected there was only a weak colocalization between eNOS phosphorylated at Ser-1177 and caveolin-1. Different from many other cell systems, human MSCs accumulate eNOS in the nucleus without an acute stimulus. eNOS constitutively phosphorylated at distinct amino acid residues is targeted to different subcellular compartments pointing to an important role of specific phosphorylation events in the life cycle of proliferating human MSCs.
authors.affiliation: Department of Anatomy I, University of Cologne, Joseph-Stelzmann-Str. 9, D-50391 Cologne, Köln, Germany. franzjosef.klinz@uni-koeln.de
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Klinz
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.forename: Timo
authors.initials: T
authors.lastname: Schinköthe
authors.forename: Stefan
authors.initials: S
authors.lastname: Arnhold
authors.forename: Biren
authors.initials: B
authors.lastname: Desai
authors.forename: Frank
authors.initials: F
authors.lastname: Popken
authors.forename: Klara
authors.initials: K
authors.lastname: Brixius
authors.forename: Robert
authors.initials: R
authors.lastname: Schwinger
authors.forename: Uwe
authors.initials: U
authors.lastname: Mehlhorn
authors.forename: Peter
authors.initials: P
authors.lastname: Staib
authors.forename: Klaus
authors.initials: K
authors.lastname: Addicks
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Bloch
mesh_terms: D021581:Active Transport, Cell Nucleus; D000954:Antigens, Surface / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D002451:Cell Compartmentation; D042783:Endothelial Cells / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology*; D008938:Mitosis; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D020033:Protein Isoforms; D021381:Protein Transport; D015854:Up-Regulation
chemical_list: D000954:Antigens, Surface; D051242:Caveolin 1; D020033:Protein Isoforms; D010768:Phosphoserine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III