Phospho-eNOS Ser-114 in human mesenchymal stem cells: constitutive phosphorylation, nuclear localization and upregulation during mitosis.
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.
Klinz
Franz-Josef
FJ
Department of Anatomy I, University of Cologne, Joseph-Stelzmann-Str. 9, D-50391 Cologne, Köln, Germany. franzjosef.klinz@uni-koeln.de
Schmidt
Annette
A
Schinköthe
Timo
T
Arnhold
Stefan
S
Desai
Biren
B
Popken
Frank
F
Brixius
Klara
K
Schwinger
Robert
R
Mehlhorn
Uwe
U
Staib
Peter
P
Addicks
Klaus
K
Bloch
Wilhelm
W
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
D000954:Antigens, Surface; D051242:Caveolin 1; D020033:Protein Isoforms; D010768:Phosphoserine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III