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First published online 25 November 2008
doi: 10.1242/jcs.037952

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Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival

Center for Cancer and Stem Cell Biology, Alkek Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA

View larger version (33K): [in this window] [in a new window]   Fig. 1. MDM2 mediates the association between nucleostemin and p53 via the central domain of MDM2 and the coiled-coil and acidic domains of nucleostemin. (A1) Triple-coimmunoprecipitation assays of HA-tagged nucleostemin (NS), Flag-tagged MDM2 and Myc-tagged p53 show that nucleostemin, MDM2 and p53 coexist in the same protein complex. (A2) Binding between nucleostemin and p53 is significantly reduced without MDM2 coexpression. (B) In vivo binding of endogenous nucleostemin and MDM2 is confirmed by coimmunoprecipitation assays, which immunoprecipitated MDM2 or nucleostemin complexes from U2OS cells. (C) MDM2 (top) and nucleostemin (bottom) deletion mutants. Gray lines indicate the deleted regions. (D) Coimmunoprecipitation assays showed that nucleostemin fails to bind MDM2 mutants with deleted I1 or AZ domains (D1), and that deleting the A domain or the BC domain of nucleostemin abolishes its ability to bind MDM2 (D2). Abbreviations: A, acidic domain; AZ, acidic/zinc finger; B, basic domain; C, coiled-coil domain; G, GTP-binding domain; I, intermediate; N, p53-binding domain; R, RING finger; Sup, supernatant; WB, western blot.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Nucleostemin binds and retains MDM2 in the nucleoplasm of living cells under nucleolar stress. (A1) In vivo interaction between nucleostemin and MDM2 was shown by the bimolecular fluorescence complementation (BiFC) approach. The Flag-tagged N-terminal (Yn) and Myc-tagged C-terminal (Yc) fragments of Venus YFP were fused to MDM2 and nucleostemin, respectively. (A2) Yn-fused MDM2 (wild-type or mutant) and Yc-fused nucleostemin (wild-type or mutant) were coexpressed in HeLa cells with a nucleolar CFP (noCFP) marker. The percentages of YFP+ cells in the CFP+ population measured by FACS are indicated in the histogram. BC and A domains of nucleostemin were deleted in NS-GI-Yc. The AZ domain and the I1 and AZ domains were deleted in MDM2M2-dAZ-Yn and M2-dIAZ-Yn, respectively. (B1) The nucleoplasmic retention time of MDM2 was measured by FLIP in HeLa cells, in which the nucleolus was bleached and the nucleoplasmic fluorescence intensity was measured. Time-sequenced images with labels indicating the bleached areas in the nucleolus (yellow circles), the measured areas in the nucleoplasm (red rectangles) and intervals between image acquisition and the first bleaching pulse (in seconds) are shown. Scale bar: 5 µm. (B2) The average FLIP rates of MDM2 were calculated from 20 cells from 2-3 independent experiments. Coexpression of wild-type or nucleoplasmic mutants of nucleostemin increased the nucleoplasmic retention time of MDM2 (P<0.0001, by Repeated Measures ANOVA). Error bars represent s.e.m. and are shown on one side (indicated by arrows) of the control and dB(256) curves. Y-axis represents the relative fluorescence index (RFI), and arrows along the top indicate bleaching pulses. (C) The role of endogenous nucleostemin in regulating the dynamic distribution of MDM2 is revealed by doxorubicin (ADR) and mycophenolic (MPA) treatment, which mobilize nucleostemin from the nucleolus to the nucleoplasm. When exposed to ADR (2 µM, 4 hours) (left panel) or MPA (40 µM, 4 hours) (right panel), the retention time of MDM2 in the nucleoplasm was increased (blue) compared to the mock-treated cells (Ctrl, black, P<0.0001). Knocking down the endogenous expression of nucleostemin (siNS) was able to reverse a significant portion of the drug-induced retention of MDM2 in the nucleoplasm (red, P<0.0001).

View larger version (58K): [in this window] [in a new window]   Fig. 3. Nucleostemin increases the protein level of MDM2 by preventing its degradation and ubiquitylation. (A) Coexpression of nucleostemin (HA-tagged) increased the protein levels of exogenously expressed MDM2 in a dose-dependent manner. (B) Knocking down the expression of nucleostemin with nucleostemin-targeting shRNAmir constructs (shNS-1 and shNS-2) decreased the amount of MDM2 protein. (C) MDM2 protein stability, with or without nucleostemin overexpression (NS vs Ctrl), was measured in H1299 cells. After cycloheximide treatment, cell lysates were collected from 0 to 4 hours at 0.5-1 hour intervals. The amount of MDM2 protein at every time point was measured in three independent experiments, adjusted based on their -tubulin amounts, and expressed as a percentage of the MDM2 at the 0 time-point. (D) Nucleostemin depletion by shNS-2 cotransfection increased the degradation of MDM2. Protein degradation assays were performed over a 50 minute window. (E) HEK293 cells were transfected with (His)6-tagged ubiquitin, MDM2 and/or nucleostemin (wild-type or mutant) plasmids as specified. Ubiquitylated MDM2 products were pulled down by Ni2+ Sepharose (His PD) and detected by anti-MDM2 (SMP14) antibody. Overexpression of wild-type nucleostemin slightly decreased the polyubiquitylation of MDM2 compared with the control sample. Overexpression of the nucleoplasmic mutants of nucleostemin (dB, G256V and dB(256)) significantly decreased the ubiquitylated products of MDM2. Sup, supernatant. (F) Conversely, depleting the endogenous nucleostemin by the nucleostemin-targeting siRNA (siNS) increased the ubiquitylation of MDM2 compared with cells treated with a scrambled siRNA (siScr).

View larger version (53K): [in this window] [in a new window]   Fig. 4. Nucleoplasmic nucleostemin competes with L23 for MDM2 binding. (A1) Triple coimmunoprecipitation of nucleostemin (HA), MDM2 (Myc) and L23 (Flag) by the indicated antibodies (IP) showed that L23 coexpression reduces the interaction between nucleostemin and MDM2. Double coimmunoprecipitation shows that L23 interacts with MDM2 (A2) but not with nucleostemin (A3). (B) The ability of nucleostemin to bind MDM2 in the presence of L23 overexpression is significantly increased by the combined mutations of dB and G256V [dB(256)]. (C) The dB(256) mutant is capable of competing with L23 for MDM2 binding in the triple coimmunoprecipitation experiments. MDM2 protein complexes (top panel) immunoprecipitated from lysates (bottom panel) containing the same amount of L23 and increasing amounts of dB(256). (D) Both nucleostemin (HA) and L23 (Flag) reside primarily in the nucleolus under normal growth conditions (Ctrl). When exposed to ADR (2 µM), actinomycin D (ActD, 0.05 µg/ml) and MPA (40 µM) for 4 hours, L23 is redistributed to the nucleoplasm, as is nucleostemin, but to a lesser extent. Distribution of nucleostemin and L23 is shown in different ADR-treated cells because of the autofluorescent property of ADR. Scale bar: 10 µm. (E) Compared with mock-treated cells, ADR and MPA significantly increased the binding between nucleostemin and MDM2. ActD had a lesser effect. (F) Proteins were extracted from cells coexpressing HA-tagged nucleostemin, Myc-tagged MDM2 and Flag-tagged L5, L11 or L23, and immunoprecipitated by anti-Myc antibody. Western blots showing that L23 competes with nucleostemin for MDM2 binding better than L5 and L11.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Nucleostemin depletion triggers G2-M arrest and apoptosis. Doxycycline (Dox)-inducible nucleostemin-knockdown (shNS) U2OS and H1299 cells and their respective controls (shScr) were created (see supplementary material Fig. S4). (A) U2OS-shNS cells displayed a 47%, 57% and 67% loss of nucleostemin protein after 4, 7 and 10 days of Dox (20 µg/ml) treatment, respectively. Nucleostemin depletion reduces MDM2 levels without changing the amount of p53. p53 transcriptional activity, as determined by the protein levels of two of its transcriptional targets, p21 and Bax, and cleaved caspase-3 (aCas3) were increased. Tub, -tubulin. (B) Population-doubling levels (PDL) and time (1/PDL in days) were measured over a 6 day period. The doubling time of Dox-treated U2OS-shNS cells was significantly prolonged compared with that in shScr cells and untreated shNS cells (black bars). The PDL of H1299 cells was unchanged by nucleostemin knockdown (grey bars). (C) Cell-cycle analyses showed decreased G1-G0 and increased G2-M and sub-G1 cell percentages in the nucleostemin-knockdown U2OS cells. (D) The percentage of prophase cells labeled with anti-phospho-Histone3 (pH3) is reduced by nucleostemin knockdown. (E) Binding between the endogenous nucleostemin and MDM2 increases in M-phase-synchronized cells.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Nucleostemin protects against drug-induced death and cell-cycle arrest. Cell-cycle analyses show that nucleostemin overexpression (NSOE, black bars) has a significant effect in protecting against ActD and MPA-induced cell death (sub-G1) and reducing the S-phase block triggered by ActD. Bars, mean of three independent duplicate experiments (n=6); error bars, s.e.m.; *P<0.01; **P<0.001.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M transition and cell survival. (A) In dividing interphase cells, nucleostemin is localized in the nucleolus (grey circle), whereas MDM2 resides in the nucleoplasm (yellow circle) and blocks the activities (red cross) of p53 by ubiquitylation (Ub) and transcriptional inhibition. (B) The nucleoli are disassembled when exposed to drugs that trigger nucleolar stress or GTP depletion. In the nucleostemin-enriched cells (left panel), nucleoplasmic translocation of nucleostemin inhibits p53 activity (red cross) by stabilizing MDM2 and by competing with L23 for MDM2 binding. In the nucleostemin-deficient cells (right panel), MDM2 is either sequestered in the nucleolus by L23 or degraded, leading to G2-M arrest and cell death. (C) Nucleolar disassembly during mitosis releases nucleostemin into the nucleoplasm or cytoplasm, allowing nucleostemin to bind and stabilize MDM2. Stabilized MDM2 inhibits p53 function and safeguards the proliferation and survival of continuously dividing cells. Mit, mitochondria; 26S, 26S proteasome.

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