Cell cycle-regulated inactivation of endothelial NO synthase through NOSIP-dependent targeting to the cytoskeleton.
Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G(2) phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G(2) phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin alpha. Mutational destruction of the NLS abolishes nuclear import and interaction with importin alpha. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G(2)-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.
Schleicher
Michael
M
Institute for Biochemistry II, University of Frankfurt Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.
Brundin
Fredrik
F
Gross
Steffen
S
Müller-Esterl
Werner
W
Oess
Stefanie
S
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D016195:G2 Phase; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D019913:Nuclear Localization Signals / Q000032:analysis / Q000378:metabolism; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases; D028901:alpha Karyopherins / Q000378:metabolism
D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C419706:NOSIP protein, human; D044767:Ubiquitin-Protein Ligases; C038753:leptomycin B