Reactive oxygen species regulate nucleostemin oligomerization and protein degradation.
Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.
Department of Medicine, Divisions of Oncology and Hematology, Stanford University School of Medicine, Stanford, California 94305, USA.
Min
M
Huang
Patrick
P
Whang
Jayanth V
JV
Chodaparambil
Daniel A
DA
Pollyea
Brenda
B
Kusler
Liwen
L
Xu
Dean W
DW
Felsher
Beverly S
BS
Mitchell
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001752:Blast Crisis / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008223:Lymphoma / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D055503:Protein Multimerization; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins
D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D016166:Free Radical Scavengers; C469949:GNL3 protein, human; D007976:Leupeptins; C489427:MYC protein, human; D009687:Nuclear Proteins; D016877:Oxidants; D061988:Proteasome Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; C470311:nucleostemin protein, mouse; D006861:Hydrogen Peroxide; D046988:Proteasome Endopeptidase Complex; D019204:GTP-Binding Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine
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