Recruitment of Phosphorylated NPM1 to Sites of DNA Damage Through RNF8-Dependent Ubiquitin Conjugates

Published OnlineFirst August 16, 2010; DOI: 10.1158/0008-5472.CAN-10-0382 Published OnlineFirst on August 16, 2010 as 10.1158/0008-5472.CAN-10-0382

Molecular and Cellular Pathobiology Cancer Research Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Ayaka Koike1,2, Hiroyuki Nishikawa3, Wenwen Wu2, Yukinori Okada1, Ashok R. Venkitaraman4, and Tomohiko Ohta1,2

Abstract Protein accumulation at DNA double-strand breaks (DSB) is essential for genome stability; however, the mechanisms governing these events are not fully understood. Here, we report a new role for the nucleophosmin protein NPM1 in these mechanisms. Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear DNA damage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiquitin ligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers in vitro. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA damage. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitin- interacting motif–like domain. Depletion of the breast cancer suppressor BRCA1 or its partner, RAP80, enhanced IR-induced NPM1 foci and prolonged persistence of the foci, possibly implicating BRCA1 in pT199-NPM1 action and dynamics. Replacement of endogenous NPM1 with its nonphosphorylable T199A mutant prolonged persistence of IR-induced RAD51 foci accompanied by unrepaired DNA damage. Collectively, our findings suggest that phosphorylated NPM1 is a novel component in DSB repair that is recruited by ubiquitin conjugates downstream of RNF8 and RNF168. Cancer Res; 70(17); OF1–11. ©2010 AACR.

Introduction the RAP80 component, which contains two ubiquitin- interacting motif (UIM) domains and directly binds to DNA double-strand breaks (DSB), such as those induced K63-linked ubiquitin conjugates (9–11). BRCA1 assists in re- by ionizing radiation (IR) or some anticancer agents, are cruitment of the BRCA2-RAD51 repair complex that executes among the most destructive and cytotoxic DNA lesions. Their HR reaction through an interaction bridged by the PALB2 repair in mammalian cells involves recruitment to DSBs of protein (12, 13). many different proteins that sense, signal, and repair these We have previously implicated nucleophosmin (NPM1) in lesions (1). The nuclear structures that reflect assembly of this cascade of events by showing that it is a candidate sub- effector proteins at or near the site of DSBs in homologous strate for the E3 activity of BRCA1 (14). NPM1 is an abundant recombination (HR) repair pathway can be detected as IR- nucleolar phosphoprotein that shuttles between the nucleus induced foci (IRIF). IRIF formation at the DSB sites is initiated and the cytoplasm. It participates in multiple cellular by transient chromatin changes followed by ATM-dependent processes, including ribosome biogenesis and transport; phosphorylation of the histone variant H2AX (γ-H2AX) and possesses antiapoptotic activity; regulates centrosome MDC1 (1–3). Phosphorylation of MDC1 recruits an E3 ubiqui- duplication; and plays crucial roles in ARF-MDM2-p53 path- ways (15). NPM1 is clinically important in hematopoietic tu- tin ligase, RNF8, which catalyzes lysine 63 (K63)-linked poly- mors because chromosomal translocations resulting in ubiquitin chains at the DSB sites together with the E2 – chimeric NPM1-kinase fusion proteins cause anaplastic lym- enzyme Ubc13 (4 6). The K63-linked polyubiquitin chains phomas (15–17). Further, mutations generating a COOH- are further enhanced by another E3 ligase, RNF168 (7, 8). terminal extension are frequently found in acute myeloid The BRCA1-Abraxas-RAP80 complex is next recruited through leukemia (18). NPM1 is also frequently overexpressed in solid tumors (15). NPM1 travels from the nucleolus to the nucleoplasm in re- Authors' Affiliations: 1Department of Translational Oncology; 2Division sponse to DNA damage, promotes p53 stabilization, and en- of Breast and Endocrine Surgery, Department of Surgery; 3Institute of Advanced Medical Science, St. Marianna University Graduate School of hances DNA repair (19–21). Moreover, proteomics analysis of Medicine, Kawasaki, Japan; and 4The Medical Research Council Cancer chromatin from γ-irradiated HeLa cells identified NPM1 as a Cell Unit, Hutchison/MRC Research Centre, Cambridge, United Kingdom DNA-damage–induced chromatin-binding protein (22). Al- Note: Supplementary data for this article are available at Cancer Research though this evidence broadly supports the notion that Online (http://cancerres.aacrjournals.org/). NPM1 is involved in DNA repair, the nature of its function Corresponding Author: Tomohiko Ohta, Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawa- in this process is very poorly characterized. Immunofluores- saki 216-8511, Japan. Phone: 81-44-977-8111; Fax: 81-44-975-1400; cence experiments show that anti-NPM1 staining does not E-mail: [email protected]. decorate sites of DNA damage after IR, leading to the as- doi: 10.1158/0008-5472.CAN-10-0382 sumption that NPM1 may function in the global context of ©2010 American Association for Cancer Research. chromatin rather than specifically exert its functions at DSBs

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(22). In this study, we show for the first time that only a frac- oligonucleotides targeting the NPM1 mRNA sequence 5′- tion of the total cellular pool of NPM1, phosphorylated on CGAAGGCAGUCCAAUUAAA-3′ or with nontargeting siRNA residue Thr199 (pT199-NPM1), is recruited to DSBs and plays (Dharmacon, D-001210-05), and analyzed 48 hours after the a critical role in the RNF8-dependent DNA repair pathway. second transfection. For Venus-IRIF studies HEK-293T cells were transfected by using the standard calcium phosphate Materials and Methods precipitation method and irradiated (5 Gy) 12 hours after transfection. Antibodies, plasmids, and purified proteins Antibodies used are shown in Supplementary Table S1. Full- Western blot analysis length human NPM1 cDNA (14) was subcloned into pcDNA4 Western blots were performed as described (25). vector or pGEX vector in frame with the COOH-terminal Myc tag or the NH2-terminal glutathione S-transferase (GST) Immunofluorescence microscopy tag, respectively. Mutants were generated by site-directed Indirect immunofluorescence labeling was performed as mutagenesis and restriction enzyme digestion. To generate described (14), with the appropriate concentration of the siRNA-insensitive NPM1-Myc constructs, the underlined antibodies (Supplementary Table S1). silent mutations (CGAAGGCTCACCAATTAAA) were intro- duced into the siRNA target sequence in the NPM1 coding GST pull-down region. For Venus-fusion proteins, BARD1 and NPM1 were GST pull-down assay was performed as described (14, 23), cloned in frame with the COOH terminus of p3FLAG- with GST fusion protein (10 μg) mixed with K63-linked ubi- – μ CMV10-Venus-N (1 172) and to the NH2 terminus of quitin polymers (3 g) containing two to seven molecules of – pcDNA3HA-Venus-C (173 238) [(gifts from Drs. Mutsuhiro ubiquitin (Ub2–7). Takekawa (Department of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Surface plasmon resonance analysis Tokyo, Japan) and Atsushi Miyawaki (Laboratory for Cell Analyses were performed using a BIAcore 3000 instrument Function and Dynamics, Advanced Technology Develop- (GE Healthcare). Purified GST-tagged NPM1 peptides or ubi- ment Group, Brain Science Institute, RIKEN, Wako-city, quitin polymers were immobilized (700–1,000 resonance Saitama, Japan)], respectively. GST-fused recombinant pro- units) on the surface of each CM5 sensor chip using a GST teins were purified as described (23). Ubiquitin and SUMO Capture kit or an amine coupling kit, respectively (GE polymers were purchased commercially (Boston Biochem). Healthcare). The analyte (either GST-NPM1 peptide, ubiquitin polymer, or SUMO polymer) was injected at different con- Cell culture centrations at a flow rate of 20 μL per minute in 20 mmol/L Cells were cultured in DMEM as previously described (23– NaCl/20 mmol/L NaPO4 (pH 5.0) containing 0.08% surfactant 25). For IR, cells were exposed to X-ray irradiation (5 Gy) and P20 (GE Healthcare). The absence of detectable nonspecific cultured for the indicated time before analyses. To analyze absorption was confirmed in this condition. An equivalent the effect of proteasome inhibitors on IRIFs, 50 μmol/L volume of each protein sample was injected over a surface MG132 (Calbiochem) or the vehicle DMSO was added to cells with no protein immobilized or with GST protein on immo- 90 minutes after IR, and cells were incubated for an addition- bilized anti-GST antibody to serve as a blank sensorgram for al 4.5 hours. For radiosensitivity, cells were irradiated and subtraction of the bulk refractive index background. For cultured in fresh medium for 4 days. Cell viabilities were an- NPM1 fragments that do not oligomerize, kinetic analyses alyzed in triplicate by using Cell Titer-Blue (Promega). were performed with a range of analyte concentrations (0, K 100, 200, 400, and 800 nmol/L). Dissociation constants ( D) siRNA and transfection were derived by using BIAevaluation version 3.1 software and siRNA oligonucleotides targeting NPM1 (M-015737-00), a 1:1 Langmuir model. BRCA1 (M-003461-01), RAP80 (M-006995-01), RNF168 (1: D- 007152-02 and 2: D-007152-03), and nontargeting control (D- Comet assay 001206-14) were purchased (Dharmacon). For RNF8 and Neutral comet assays were performed using Trevigen's Ubc13 siRNA, oligonucleotides used in a previous report CometAssay kit (4250-050-K, Trevigen). Mean tail moment (5), which target the following sequences, were synthesized was analyzed by the TriTek CometScore Freeware program (sense strand): RNF8 (5′-AGAAUGAGCUCCAAUGUAUUU- with 100 cells per sample. 3′) and Ubc13 (5′-GCACAGUUCUGCUAUCGAUUU-3′). RNA duplexes (final concentration 50 nmol/L) were transfected Results into the cells by using Lipofectamine 2000 (Invitrogen). For the NPM1 depletion and mutant add-back experiment, U2OS NPM1 phosphorylated on residue Thr199 forms IRIF cells were first transfected with pcDNA4 plasmids encoding Although several lines of evidence support the notion that siRNA-insensitive wild-type or T199A mutant NPM1-Myc. NPM1 could have a role in the DSB repair pathway, NPM1 Cells stably expressing the proteins were selected with has not been observed at sites of DNA damage. However, be- 40 μg/mL zeocin and maintained in medium containing cause NPM1 is an abundant protein, only a small fraction of 20 μg/mL zeocin. Cells were then transfected twice with siRNA the total cellular pool of NPM1 protein may participate in the

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pathway. Supporting this idea, we found that phosphorylated IRIF formation (Supplementary Fig. S4C–E). These results sug- NPM1, pT199-NPM1, is a candidate for this specific function. gest that pT199-NPM1-IRIF formation is involved in the DSB- HeLa and U2OS cells were irradiated. pT199-NPM1 foci be- induced RNF8 cascade and that it is an event downstream of came clear within 1 hour to several hours after IR treatment RNF8-Ubc13– and RNF168-Ubc13–induced ubiquitination. and colocalized with γ-H2AX foci, conjugated ubiquitin foci, and BRCA1 foci (Fig. 1A; Supplementary Fig. S1), supporting BRCA1-IRIF are not required for pT199-NPM1-IRIF the idea that pT199A-NPM1 is recruited to DSB sites. pT199- formation NPM1-IRIF formation was detected in all cell lines tested, in- To further clarify the step in the cascade at which pT199- cluding HeLa, U2OS, MCF7, T47D, 293T, and MCF10A normal NPM1 is recruited, we next inhibited BRCA1-IRIF by siRNA human mammary epithelial cells. Inhibition of NPM1 expres- knockdown of either BRCA1 or RAP80. Individual siRNA sion with siRNA significantly decreased the amount of foci treatment successfully inhibited expression of BRCA1 and recognized by anti–pT199-NPM1 immunostaining (Fig. 1B; RAP80 (Fig. 3A), resulting in depletion of BRCA1-IRIF forma- Supplementary Fig. S1E), ensuring that the foci are indeed tion in both cases (Fig. 3B). However, neither BRCA1 nor composed of phosphorylated NPM1. We also examined the RAP80 depletion inhibited pT199-NPM1-IRIF formation kinetics of steady-state level of pT199-NPM1 after IR in each (Fig. 3C). During these experiments, we noticed that pT199- cellular fraction by immunoblot (Supplementary Fig. S2). NPM1-IRIF formation was enhanced by BRCA1 or RAP80 pT199-NPM1 in the cytoplasm and the nuclear extract was depletion. To further examine the effect, the kinetics of foci dramatically reduced within 1 hour after IR. On the contrary, formation were monitored following IR treatment. Six hours pT199-NPM1 in the chromatin fraction was increased 30 min- after IR exposure, BRCA1- and RAP80-depleted cells exhib- utes after IR, supporting the idea that a part of pT199-NPM1 ited higher numbers of pT199-NPM1-IRIF per cell than con- is incorporated at the DSB sites. trol cells (Fig. 3C). The number continued to increase until We previously found that NPM1 interacts with BRCA1- 12 hours post-IR, at which time foci in control cells were BARD1 (14). To examine whether NPM1 interacts with this hardly detectable (Fig. 3C and D). These results indicate that complex at sites of DNA damage, we cotransfected plasmids pT199-NPM1 is recruited to the DSB sites independently of encoding fusions of NPM1 to the COOH terminus of the fluo- the RAP80-Abraxas-BRCA1 complex; however, the complex is rescent protein Venus (NPM1-VC) and BARD1 to the NH2 required for normal turnover of NPM1 at the sites. terminus of Venus (VN-BARD1). Successful assembly of the Venus protein as detected by fluorescence would indicate NPM1 interacts with K63-linked ubiquitin specific interaction of NPM1 with BARD1 (Fig. 1C). Whereas conjugates in vitro untreated cells only exhibited weak fluorescence in the nu- We next tested the possibility that pT199-NPM1 binds di- cleolus, nuclear fluorescent foci became apparent after IR rectly to the ubiquitin conjugates in a manner similar to and colocalized with BRCA1 foci or γ-H2AX foci (Fig. 1C RAP80 (10). Notably, phosphomimic T199D mutant of GST- and D). This suggests that NPM1 interacts with the NPM1, but not the wild-type or T199A, interacted with K63- BRCA1-BARD1 complex at sites of DNA damage. Fluorescent linked polyubiquitin (Ub2–7), preferring polymers containing Venus-IRIF formation significantly decreased when VN- at least three ubiquitin molecules (Fig. 4A). Further, surface BARD1 was substituted with nonfused VN or when NPM1- plasmon resonance (SPR) analyses showed that T199D- VC was substituted with MEK-VC (Fig. 1D; Supplementary NPM1 directly interacted with K63-linked tetraubiquitin Fig. S3). and its steady-state response was higher than wild-type (Fig. 4B). Importantly, substitution of K63-linked tetraubiqui- Inhibition of conjugated ubiquitin-IRIF abolishes tin with K48-linked tetraubiquitin significantly decreased the pT199-NPM1-IRIF formation response (Fig. 4B). The colocalization of pT199-NPM1-IRIF with repair pro- To determine the site responsible for interaction with the teins prompted us to test whether inhibition of RNF8- and ubiquitin conjugates, we constructed several NPM1 frag- RNF168-dependent ubiquitination at DSB sites affects the fo- ments that contained T199D (Fig. 5A). Human NPM1 is com- ci formation. We first used the 26S proteasome inhibitor posed of several domains, including an NH2-terminal core MG132. Consistent with a previous report (4), inhibition of domain that is responsible for oligomer formation and three the 26S proteasome trapped conjugated ubiquitinated pro- acidic tracts (A1, A2, and A3; Fig. 5B; refs. 15, 26, 27). Whereas ducts in the cytosol and impaired ubiquitin-IRIF (Fig. 2A). fragments composed of residues 152 to 259 or 1 to 212 of Importantly, this was accompanied by the disappearance of NPM1-T199D interacted with the K63-linked ubiquitin poly- pT199-NPM1-IRIF, whereas γ-H2AX-IRIF remained intact mer, the fragment composed of residues 189 to 259 that lacks (Fig. 2A; Supplementary Fig. S4A). To further determine the the third acidic tract (A3; 161–188) did not (Fig. 5A). The re- role of RNF8-Ubc13–mediated ubiquitination in pT199- sults suggest an interaction between the ubiquitin conjugates NPM1-IRIF formation, we inhibited the expression of RNF8 and the region of NPM1 located between residues 152 and or Ubc13 with siRNA knockdown (Fig. 2B). Depletion of 212, which include the A3 domain and the T199 phosphory- RNF8 and Ubc13 resulted in inhibition of BRCA1-IRIF, lation site (Fig. 5B). The results also indicate that the inter- conjugated ubiquitin-IRIF, and pT199-NPM1-IRIF, whereas action does not require oligomerization of NPM1. γ-H2AX-IRIF remained intact (Fig. 2C; Supplementary We noticed that there is a region relatively similar to a Fig. S4B). Depletion of RNF168 also inhibited pT199-NPM1- UIM motif (UIM-like; UIML) that is adjacent to or overlaps

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Figure 1. IRIF formation of NPM1. A, HeLa cells were untreated (−) or treated with 5 Gy IR, incubated for 6 h, and immunostained with anti–pT199-NPM1 and anti–conjugated-ubiquitin (FK2) antibodies. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). B, U2OS or HeLa cells transfected with control (siCTR) or NPM1-targeting (siNPM1) siRNA oligonucleotides were immunoblotted with NPM1 antibody (left panels) and analyzed for IRIF with anti–pT199-NPM1 and anti-γH2AX antibodies (right panels). C and D, NPM1 interacts with BARD1 at sites of DNA damage. C, the scheme illustrates the principle used to detect the interaction between NPM1-BARD1 and the Venus fluorescent protein. HEK-293T cells transfected with NPM1-VC (Venus-C) and VN (Venus-N)–BARD1 were left untreated (−) or irradiated (IR) and examined for Venus-IRIF and BRCA1 immunostaining. D, HEK-293T cells treated as in C or with nonfused VN (bottom) were examined for colocalization of Venus-IRIF with BRCA1- or γ-H2AX-IRIF.

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Figure 2. pT199-NPM1-IRIF formation is impaired by perturbation of DNA-damage–associated ubiquitin conjugates. A, HeLa cells were treated with MG132 or DMSO and examined for IRIF with the indicated antibodies. B and C, HeLa cells transfected with either control, RNF8-, or Ubc13-specific siRNA were subjected to immunoblot with anti-RNF8 antibody or to reverse transcriptase-PCR with Ubc13-specific primers, respectively (B), and analyzed for IRIF with the indicated antibodies (C). with the A3 tract in the ubiquitin-binding region (Fig. 5B with T199D, point mutations of residues in UIML were intro- and D). We therefore investigated whether this region inter- duced in the GST-NPM1-A3+UIML fragment (Supplementary acts with ubiquitin conjugates. GST-tagged fragments of Fig. S5A), and dissociation constants (KD) were determined T199D-NPM1, A3+UIML (152–212), or UIML (181–212) were (Fig. 5D; Supplementary Fig. S5E; Supplementary Table S2). immobilized onto a biosensor chip (Supplementary Fig. S5A), Some mutations of the residue conserved in UIM motif, in- and K63-linked tetraubiquitin was injected as an analyte. cluding e182-5A (1.57 μmol/L), A190G (1.35 μmol/L), and Whereas the A3+UIML fragment interacted with tetraubiqui- D198A (2.12 μmol/L), significantly decreased the affinity, tin, the UIML fragment did not (Fig. 5C), consistent with the whereas S195D (0.27 μmol/L), a mutation of another con- pull-down assays that required the A3 tract for interaction. served residue, did not. A186G (0.31 μmol/L), which retains Using the A3+UIML fragment of NPM1, which contains on- the hydrophobic status, did not affect the affinity. Some ly a single CDK-phosphorylation site, T199 (Fig. 5B), we next mutations of the residue that are not conserved in UIM tested if phosphorylation of T199, instead of phosphomimic motif, including E187A (1.03 μmol/L), E188A (1.13 μmol/L), mutant T199D, indeed contributes to the binding. Supporting P191A (1.25 μmol/L), and V192D (2.05 μmol/L), also de- the phosphorylation-dependent interaction, CDK2/cyclin A– creased the affinity. In vitro binding experiments indicate phosphorylated pT199-A3+UIML (Supplementary Fig. S5B) that the A3 tract, UIML domain, and phosphorylated T199 bound to the ubiquitin polymer with significantly higher af- are all required for high-affinity interaction with K63-linked finity than the unphosphorylated A3+UIML (Fig. 5C; Supple- ubiquitin polymers, and that molecular mechanism for ubi- mentary Fig. S5C). The dissociation constant of binding of quitin interaction of NPM1-UIML could be different from K63-linked tetraubiquitin to pT199-A3+UIML (KD;0.35 conventional UIM. μmol/L) was 4.4-fold greater than that to unphosphorylated A3+UIML (1.55 μmol/L). Importantly, substitution of the Substitution of endogenous NPM1 with the T199A K63-linked tetraubiquitin with K48-linked tetraubiquitin or mutant causes failure to repair DNA damage monoubiquitin abolished the binding to pT199-A3+UIML To observe phenotypes in response to DNA damage specif- (Supplementary Fig. S5C), again supporting the linkage- ically due to loss of pT199-NPM1, we expressed an siRNA- specific interaction. SUMO polymers also did not interact insensitive, wild-type, or nonphosphorylable T199A mutant with the NPM1 fragments (Supplementary Fig. S5D). of NPM1 in NPM1-depleted cells and studied IRIF formation. We next evaluated the contribution of each residue within The desired, engineered expression of NPM1 was confirmed the UIML to K63-linked ubiquitin polymer binding. Together by Western blot (Fig. 6A, lanes 3 and 5). The wild-type cells

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exhibited pT199-NPM1-IRIF, whereas T199A cells did not DSB sites but is required for the normal process of the repair (Fig. 6A; Supplementary Fig. S6), supporting the presumption after recruitment of these proteins. that the exogenous wild-type NPM1 but not its T199A mutant The persistent retention of RAD51- and BRCA1-IRIF in can replace endogenous NPM1 at the sites of DNA damage. T199A cells raised the question of whether DNA damage is Using these cells, we tested whether inhibiting NPM1 T199 left unrepaired. Consistent with this possibility, the number phosphorylation affects RAD51-IRIF, a marker for HR repair of γH2AX-IRIF was higher in T199A cells than that in wild- acting downstream of the RNF8-RAP80-BRCA1 cascade. The type cells (Supplementary Fig. S8A). We further performed number of RAD51-IRIF in T199A was approximately the same neutral comet assay to directly measure DSBs (Fig. 6C; Sup- as that in wild-type cells until 6 hours after IR exposure (Fig. plementary Fig. S8B). Immediately after irradiation, approxi- 6B, top). However, a dramatic difference was observed 12 to mately the same amount of DNA fragments were generated 18 hours post-IR. Whereas the number of RAD51-IRIF in wild- in wild-type and T199A cells, an observation that was noted type cells decreased to approximately half the level observed until 6 hours post-IR. However, T199A cells exhibited an at 6 hours post-IR, the foci in T199A cells continued to in- increased amount of DNA fragments at 12 and 24 hours crease until 18 hours post-IR (Fig. 6B, top). U2OS and HeLa post-IR, whereas wild-type cells repaired a majority of the cells simply transfected with NPM1-specific siRNA also exhib- damage by that time. These results suggest that retention ited kinetics similar to that of T199A cells (Fig. 6B, middle and of phosphorylated NPM1 to sites of DNA damage is required bottom). Similar results were observed for BRCA1-IRIF (Sup- for DSB repair. The increased DNA damage may reflect newly plementary Fig. S7). These results suggest that phosphorylat- generated DSBs due to continuous DNA replication, which ed NPM1 is dispensable for recruiting BRCA1 and RAD51 to can convert IR-induced single-strand breaks to DSBs. In

Figure 3. BRCA1 or RAP80 depletion leads to enhanced formation and prolonged persistence of pT199-NPM1-IRIF. A and B, HeLa cells transfected with control, BRCA1-specific, or RAP80-specific siRNA were immunoblotted with the indicated antibodies (A) and examined for IRIF with anti-BRCA1 and anti-γH2AX antibodies (B). C, cells were then examined for pT199-NPM1-IRIF. The time course of pT199-NPM1-IRIF formation per cell is shown at the indicated time after 5 Gy IR. Means of 100 cells with SD at each time point are shown. D, representative data of cells 12 h post-IR are shown with pT199-IRIF (red) and DAPI (blue) stains.

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Figure 4. Phosphomimic T199D mutant of NPM1 interacts with K63-linked ubiquitin conjugates. A, a mixture of K63-linked ubiquitin polymers (Ub2–7) was incubated with wild-type (WT) GST-NPM1 (B23.1), T199D mutant, or T199A mutant (B23.2). Immunoblot was performed with anti-ubiquitin antibody

(P4D1) after GST pull-down (left). The Ub2–7 input (15%) was also loaded. The GST-tagged proteins used in the reactions were stained with Coommasie brilliant blue (CBB, right). *, GST-NPM1 peptides. B, binding curves of wild-type or the T199D mutant of GST-NPM1 obtained by SPR analyses with K63- or K48-linked tetraubiquitin peptides. The concentrations of analyte injected (GST-NPM1) were 50, 100, 200, and 400 nmol/L.

addition, the different amounts of DNA damage could be due It is intriguing that NPM1 recruitment is through a newly to different proportions of S-phase cells between wild-type identified UIML motif in cooperation with an NH2-terminal and T199A cells. However, cell cycle analyses showed approx- acidic tract (A3) and COOH-terminal T199 phosphorylation. imately the same proportions of the S-phase fraction in wild- The UIM motif is an 18- to 21-residue sequence composed type and T199A cells before and after IR (Supplementary of highly conserved alanine and serine residues flanked by Fig. S9). On the other hand, T199A cells after IR showed a conserved acidic patches (Fig. 5D; refs. 31–33). A single UIM higher proportion of apoptotic cells, which may reflect a part is capable of binding to a single ubiquitin molecule, al- of the increased DNA fragments observed in T199A cells. though the affinity is relatively low in general (34). In the The significant increase in unrepaired DNA damage ob- case of RAP80, tandem UIMs greatly increase the affinity served in T199A cells prompted us to test whether it in- to K63-linked diubiquitin because each UIM interacts with creases the sensitivity of cells to IR (Fig. 6D). The viability each ubiquitin molecule (35, 36). However, the UIML motif of T199A cells after IR was lower than that of wild-type or found in NPM1 is significantly different from the classic control cells at all IR doses examined, although the effect UIM in that it possesses an additional two residues between was relatively mild. On the other hand, NPM1 depletion did the NH2-terminal acidic domain and the central alanine and not dramatically affect radiosensitivity. serine residues. Indeed, neither UIML or A3+UIML interacted with single ubiquitins. This may indicate that this Discussion motif interacts with ubiquitin conjugates in a manner different from UIMs. In addition to the UIML domain, the In this study, we show that NPM1 is recruited to DSBs in acidic A3 tract may help to increase the affinity by interact- a manner dependent on RNF8/RNF168-mediated ubiquiti- ing with another ubiquitin molecule in the K63-linked nation. Unlike BRCA2 and RAD51, the recruitment of chain. Although the UIML domain possesses a COOH- NPM1 is not through the BRCA1 complex. Instead, our data terminal acidic residue (D198), our results indicate that suggest that NPM1 is recruited through direct interaction phosphorylation of T199 is required for the full affinity of with K63-linked ubiquitin conjugates. In addition to interaction with ubiquitin conjugates, probably by enhanc- RAP80, several other proteins are also recruited through in- ing the acidity of the COOH-terminal patch of the motif. To teraction with K63-linked ubiquitin conjugates at DSB sites. our knowledge, this is the first example for a ubiquitin- These include RNF168, which possesses a motif interacting binding module composed of phosphorylation. with the ubiquitin domain (7, 8, 28), and RAD18, another E3 The recruitment of NPM1 by the K63-linked ubiquitin ligase that binds to ubiquitin through its zinc finger domain conjugates, which also directly recruits the RAP80-Abraxas- (29). Components of the RAP80-BRCA1 complex (called the BRCA1 complex (10), together with NPM1-BARD1 interac- BRCA1 A complex), including Abraxas, BRE, BRCC36, and tion at the sites of DNA damage, raises the possibility that NBA1, were also shown to directly interact with ubiquitin both NPM1 and BRCA1-BARD1 are recruited together in conjugates in vitro through MPN, Uev, MPN+, and VWA do- the same ubiquitin-containing complex. This is interesting mains, respectively (30). Therefore, the complex formation from the context that NPM1 interacts with BRCA1-BARD1 of repair proteins at DSB sites depends on ubiquitin conju- and serves as an efficient substrate for the E3 activity (Sup- gates in multifaceted ways. Our results suggest that NPM1 plementary Fig. S10; ref. 14). The reciprocal requirement of is one such protein. BRCA1 and pT199-NPM1 for normal turnover of each protein

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at the sites of DNA damage could be ascribed to the func- in the S-phase of the normal cell cycle (37–39). In response tional interaction of the two proteins. to IR treatment, pT199-NPM1 disappeared from soluble T199 phosphorylation of NPM1 is mediated by the cyclin- fractions likely due to inhibition of CDK by checkpoint dependent kinases CDK1 and CDK2. The phosphorylation activity. However, pT199-NPM1 instead appeared in the levels are highest during mitosis, but they are also expressed chromatin fraction. This suggests that pT199-NPM1 resides

Figure 5. Domains and modification required for the interaction of NPM1 with K63-linked ubiquitin conjugates. A, the interaction of K63-linked Ub2–7 with truncated mutants of GST-NPM1-T199D was examined as in Fig. 4A. B, domain structure of NPM1 and the sequence of residues 152 to 212 that contain A3 and UIML domains. NES, NLS, NuLS, and P represent nuclear exporting signal, nuclear localization signal, nucleolar localization signal, and phosphorylation site, respectively. C, SPR analyses of K63-linked tetraubiquitin peptides binding to immobilized A3+UIML or UIML fragment of GST-NPM1-T199D (left), and to immobilized phosphorylated (pT199) or unphosphorylated A3+UIML fragment of wild-type GST-NPM1 (right). D, the dissociation constants of binding of K63-linked tetraubiquitin to GST-NPM1 (A3+UIML) containing the indicated mutation. The corresponding sequence alignment of the UIML motif of NPM1 is indicated at the bottom together with the conventional UIM motif. ϕ, e, and x represent hydrophobic, acidic, and any amino acid residue, respectively.

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Figure 6. Depletion of pT199-NPM1 prolongs the persistence of RAD51-IRIF accompanied by unrepaired DNA damage. A, U2OS cells expressing either wild-type or T199A mutant of Myc-tagged, siRNA-resistant NPM1 were depleted of endogenous NPM1 with siRNA (lanes 3 and 5) or treated with control siRNA (lanes 2 and 4). The expression of endogenous and exogenous NPM1 was monitored by immunoblotting with anti-NPM1 antibody (left). Lane 1, parental U2OS cells. Cells from lane 3 (wild-type) and lane 5 (T199A) were analyzed for pT199-NPM1-IRIF (right). B, the cells from A (top) or U2OS (middle) and HeLa (bottom) cells depleted of total NPM1 were examined for RAD51-IRIF. The time courses of number of RAD51-IRIF per cell are shown at the indicated time after 5 Gy IR (left). Means of 100 cells with SD at each time point are shown. Representative data of RAD51-IRIF in the cells 18 h post-IR are shown with DAPI stain (right). C, cells from A were untreated or irradiated with 5 Gy, collected immediately (0 h) or at the indicated time after IR, and analyzed for the presence of DSB by using the neutral comet assay. The mean tail moment of comet assays is shown with SDs. D, U2OS cells from A (WT and T199A) and cells simply transfected with control or NPM1-targeting siRNA oligonucleotides were irradiated at the indicated doses, and cell viabilities were determined 4 d after irradiation.

in S-phase nucleoplasm before DNA damage, and assembly components (39) in addition to its role in binding to ubiqui- of K63-linked ubiquitin conjugates at DSB sites triggers re- tin conjugates. cruitment of pT199-NPM1 from the nucleoplasm. Phosphor- We showed that NPM1 is dispensable for recruitment of ylation contributes to NPM1 dissociation from nucleolar RAD51 but required for DNA damage repair, presumably at

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Koike et al.

a late stage of the repair cascade. One possible feature of However, selective inhibition of pT199-NPM1 resulted in in- NPM1 that may contribute in this process is its histone chap- crease of radiosensitivity, although the effect was modest. erone activity. Structural and biochemical analyses revealed The difference may come from selective inhibition of DSB re- that NPM1 is a decameric histone chaperone that interacts pair and conserved apoptotic pathway in T199A cells. Dual with core histones (26, 27, 40, 41). NPM1 may deliver histones roles of NPM1 on DSB repair and apoptosis may affect tu- to DNA in collaboration with ATP-utilizing chromatin remo- morigenesis and chemosensitivity of a variety of cancers, in- deling factors. From this perspective, it could be interesting cluding leukemia and breast cancer. that some histone chaperones and chromatin assembly factors, such as Spt6, RSF1, CAF1A, and NPM2, possess mul- Disclosure of Potential Conflicts of Interest tiple acidic tracts and possible CDK-phosphorylation sites. In general, NPM1 inhibits apoptosis and enhances cell pro- No potential conflicts of interest were disclosed. liferation (15). Compatibly, NPM1−/− embryos show high le- Acknowledgments vels of apoptosis, accompanied by stabilization of p53 (42, 43). However, this effect can be explained by two mechan- We thank H. Gomi and members of the Department of Radiology for isms: (a) a response to DNA damage caused by the absence support in IR experiments, and H. Asakawa for assistance in protein of NPM1 and (b) the direct function of NPM1 to inhibit the purification. ARF-MDM2-p53 pathway (15, 43). Our results suggest that the pT199-NPM1–dependent HR repair is involved in the first Grant Support

mechanism. A previous report (44) and our results showed Ministry of Education, Science, Sports, Culture and Technology of Japan. that depletion of total NPM1 had no effect on survival after The costs of publication of this article were defrayed in part by the payment IR, probably reflecting the multiple functions of NPM1 in ad- of page charges. This article must therefore be hereby marked advertisement in dition to DSB repair. Although overexpression of NPM1 inhi- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bits ARF, depletion of NPM1 also inhibits ARF because NPM1 Received 01/30/2010; revised 07/07/2010; accepted 07/08/2010; published binds to and protects ARF from degradation (43, 45, 46). OnlineFirst 08/16/2010.

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Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Ayaka Koike, Hiroyuki Nishikawa, Wenwen Wu, et al.

Cancer Res Published OnlineFirst August 16, 2010.

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