Introduction

Oncogene-induced senescence (OIS) is an important cellular response for protection against cancer development1,2,3,42, HIPK227, and pVHL34 have been identified. Therefore, we hypothesized that ATM is ubiquitinated by WSB1, which results in its downergulation. To test this hypothesis, we first examined the ability of WSB1 to interact with ATM. The association of endogenous ATM with WSB1 was detected via co-immunoprecipitation, and this binding was increased after MG132 treatment, which increased both ATM and WSB1 levels (Figure 3A). To identify the regions of ATM involved in binding WSB1, we performed glutathione S-transferase (GST) pull-down experiments using 8 overlap** GST-ATM fragments spanning full-length ATM43. WSB1 bound strongly to GST-ATM-5 (a.a. 1 690-2 120) and weakly to GST-ATM-8 (a.a. 2 680-3 056, encompassing the kinase domain; Figure 3B). We further determined whether WSB1 could regulate ATM ubiquitination. We found that WSB1 overexpression increased ATM ubiquitination (Figure 3C). In addition, in cells depleted of WSB1, ATM stability increased (Figure 3D). Furthermore, p53 stability, which is regulated by ATM, was also increased in WSB1-depleted cells. However, we did not find WSB1 to regulate ATR or DNA-PK stability, nor did it interact with ATR or regulate ATR expression (Figure 3D-3F). These results suggest that WSB1 regulates ATM levels through the ubiquitin-proteasome pathway.

Figure 3
figure 3

WSB1 regulates ATM ubiquitination and degradation. (A) Co-immunoprecipitation (Co-IP) of endogenous ATM and WSB1 from extracts of HEK 293T cells. (B) Purified WSB1 were incubated with GST or equal amounts of GST-ATM fragments coupled to GSH sepharose. Proteins retained on sepharose were then blotted with the indicated antibodies. (C) Cells were transfected with the indicated constructs and then treated with MG132. Ubiquitinated proteins were pulled-down under denaturing conditions by Ni-NTA agarose and analyzed by immunoblot. (D) HEK 293T cells stably expressing control shRNA or WSB1 shRNA (#1 and #2) were treated with CHX (0.1 mg/ml) and harvested at the indicated times. Cell lysates were then blotted with the indicated antibodies (left). Data (left) were quantified and normalized relative to the β-actin level using ImageJ program (right). **P < 0.01 and ***P < 0.001 versus control cells by one-way ANOVA. (E) Co-IP of WSB1 and ATM or ATR from extracts of HEK 293T cells. (F) Cells were transfected with WSB1 shRNAs and ATM or ATR levels were examined by immunoblot. (G, H) Cells were transfected with the indicated constructs and then treated with MG132. Ubiquitinated proteins were pulled-down under denaturing conditions by Ni-NTA agarose and analyzed by immunoblot. (I) ATM KO MEFs were transfected with the indicated constructs and ATM levels were then examined by immunoblot (top). Quantification of ATM protein expression levels are shown in top panel (bottom). *P < 0.05 and ***P < 0.001 versus control cells by one-way ANOVA. (J) Cells were infected (or transfected by electroporation) with the indicated viruses (or constructs) and analyzed for senescence (top, SA-β-gal staining; bottom, quantification of senescent or transformed cells).

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As some ATM ubiquitination sites were revealed by mass spectrometry analysis (http://www.phosphosite.org), we analyzed whether these sites were ubiquitinated by WSB1. Of them, mutation of either K1323 or K2025 to arginine (R) substantially reduced ATM ubiquitination (Figure 3G), suggesting that these two sites might be major sites of ubiquitination mediated by WSB1. As shown in Figure 3H, double mutation of both K1323 and K2025 residues (2KR) largely abolished ATM ubiquitination induced by WSB1 overexpression. We therefore predicted that the 2KR mutant would be resistant to WSB1-mediated downregulation. To test this hypothesis, we stably expressed Flag-tagged WT ATM or ATM mutants (K1323R, K2025R, K1323R/K2025R) in ATM−/− immortal MEFs (Figure 3I). The level of WT ATM was dramatically decreased by WSB1 overexpression. The expression of single KR mutants of ATM could also be decreased by WSB1 expression, although to a lesser extent than WT ATM. Importantly, the expression of 2KR was not affected by WSB1 expression (Figure 3I). We next tested whether reintroducing 2KR, but not WT ATM can confer resistance to WSB1-dependent escape of OIS. Both control cells and MEFs expressing WT ATM could be transformed by H-RasV12 (Figure 3J). However, cells expressing 2KR were senescent and resistant to transformation by H-Ras. Furthermore, WSB1 knockdown inhibited cell transformation induced by H-RasV12 in cells expressing WT ATM, and had no additional effect in cells expressing the 2KR mutant. All of the above results indicated that K1323 and K2025 are major ATM ubiquitination sites regulated by WSB1, and ubiquitination of these residues leads to ATM degradation.

The SOCS domain of WSB1 is required for ATM interaction and ubiquitination

We next determined how WSB1 interacts with ATM. WSB1 contains several WD40 repeats and a SOCS domain42. WD40 repeats participate in various cellular functions, and the SOCS box is involved in protein degradation by the Elongin-Cullin-SOCS-box protein ubiquitin ligase complex44,45,46. To determine the region of WSB1 that is required for its interaction with ATM, we transfected Myc-tagged WSB1-deletion mutants into HEK 293T cells and performed co-immunoprecipitation. We found that the C-terminal WD40 repeats (WD 6-7) and the SOCS domain of WSB1 were both required and sufficient for ATM interaction (Figure 4A and 4B). We further found that downregulation of ATM by WSB1 was dependent on the SOCS domain (Figure 4A and 4C). Consistent with these results, we found that ubiquitination of ATM by WSB1 required the SOCS domain (Figure 4D). In addition, ATM was more stable in cells expressing the WSB1 mutant lacking the SOCS domain (Figure 4E). Together, these results indicate that WSB1 interacts with ATM via its WD40 repeats and SOCS domain and regulates ATM ubiquitination and degradation through the SOCS domain.

Figure 4
figure 4

WSB1 interacts with ATM via its WD40 repeats and SOCS domain and regulates ATM ubiquitination through the SOCS domain. (A) Diagrams of WT WSB1 and corresponding deletion mutants (ΔWD 1-3, ΔWD 1-5, ΔSOCS, and Δ6-SOCS) used in Co-IP experiments with ATM. Plus symbols indicate binding ability between different WSB1 mutants with ATM (left) or the degradation strength of the WSB1 mutants on ATM stability (right). (B) Cells were transfected with indicated plasmids and then treated with MG132. Cells were collected for immunoprecipitation (IP)-immunoblot analysis. (C) Cells were transfected with indicated plasmids and the ATM levels were examined by immunoblot. (D) Cells were transfected with the indicated constructs and then treated with MG132. Ubiquitinated proteins were pulled-down under denaturing conditions by Ni-NTA agarose and analyzed by immunoblot. S.E. indicates short exposure; L.E., long exposure. (E) Cells were treated with CHX (0.1 mg/ml) and harvested for different hours. ATM levels were then examined by immunoblot (left). Right panel shows quantification of ATM levels. The results represent the means (± SE) of three independent experiments performed in triplicate. **P < 0.01 and ***P < 0.001 versus control cells by one-way ANOVA. Data (left) were quantified and normalized relative to the β-actin level using ImageJ program (right). (F, G) Primary MEFs were infected with the indicated constructs and were analyzed for senescence by SA-β-gal staining assay (F) and cell-proliferation assay (G). Scale bar, 10 μm. Yellow arrows indicate SA-β-gal stained cells; red arrows, transformed cells; white arrow, death cells. The results represent the means (± SE) of three independent experiments performed in triplicate. **P < 0.01 and ***P < 0.001 versus control cells by one-way ANOVA. (H) Cells as in F were collected for immunoblot analysis. (I, J) Primary MEF cells as in F were applied on 3-D organoid cultures system. Schematic of the experiments (I) and sphere number (J) in 3-D organoid culture. ****P < 0.0001 versus control cells by one-way ANOVA. (K) IMR-90 ER:Ras-inducible cells were infected with indicated viruses, and H-Ras was inducted by 4-OHT. Cells were analyzed for 'cytokine' mRNAs by real-time PCR analysis.

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We reasoned that WSB1-induced ubiquitination and degradation of ATM could facilitate cellular transformation by oncogene through the suppression of DDR. Given the important function of the WSB1 SOCS domain in mediating ATM degradation, we investigated how the WSB1 mutant lacking the SOCS domain affects OIS. We infected MEFs with viruses encoding both H-RasV12 and WSB1 (WT or ΔSOCS) and examined OIS and cellular transformation. Consistent with Figure 1, co-expression of oncogenic Ras and WSB1 promoted the escape from OIS and cell transformation (Figure 4F and 4G). Deletion of the SOCS domain abolished the ability of WSB1 to promote the escape from OIS, cell transformation, and abnormal cell proliferation (Figure 4F and 4G). ATM levels were decreased in cells expressing WT WSB1 but not in cells expressing mutant WSB1 (ΔSOCS; Figure 4H). In 3-D culture systems, we obtained similar results. WT WSB1, but not ΔSOCS, together with H-RasV12 expression, promoted sphere formation (Figure 4I and 4J). Since senescence-associated secretion phenotype (SASP) is a major characteristic of OIS3,47, we next examined the impact of WSB1 on SASP. We found that overexpression of WT WSB1 suppressed the expression of key SASP factors such as IL-6, IL-8, and IL-1β during OIS after H-RasV12 induction in IMR-90 ER:Ras cells (Figure 4K). However, WSB1 mutant lacking the SOCS domain did not affect SASP gene expression. These results together with Figure 2 suggest that one major mechanism by which WSB1 promotes the escape from OIS thus leading to cellular transformation is through inducing ATM ubiquitination and its subsequent degradation.

WSB1 phosphorylation by CDKs is required for its monomerization and activity

We next sought to determine the molecular mechanisms that regulate WSB1. For several E3 ligases, such as MDM2 and TRAF6, dimerization or oligomerization is involved in the regulation of activity48,43. WSB1 and ATM mutations were generated by site-directed mutagenesis (Stratagene).

Transient transfection, viral infection, and stable transduction

shRNAs or viral vectors were infected using Lipofectamine 2000 reagent (Invitrogen). Human WSB1 and mouse WSB1 shRNAs were obtained from Sigma-Aldrich and Open Biosystems.

WSB1 shRNA (human): Open Biosystems

5′-GCTGTTGACAGTGAGCGCGGAGTTTCTCTCGTATCGTATTAGTGAAGCCACAGATGTAATACGATACGAGAGAAACTCCATGCCTACTGCCTCGGA-3′

5′-GCTGTTGACAGTGAGCGCGCTGTAAAGTGCAAGGAAATTTAGTGAAGCCACAGATGTAAATTTCCTTGCACTTTACAGCATGCCTACTGCCTCGGA-3′

WSB1 shRNA (Mouse): Sigma-Aldrich

5′-ACATGAGCTGCTGCTATATAT-3′

5′-GCTTACTCCTTGTATCAGCTT-3′

ATM shRNA (Mouse): Sigma-Aldrich

5′-CCGTGGAGATTTCTCAATCTT-3′

5′-CCTCCAATTCTTCAGCGTAAT-3′

5′-GCTGAGACAAATAATGTCTTT-3′

5′-CGATGGAAGTTATGCGGAGTT-3′

5′-CCACCATATTTGGACAGGAAT-3′

For transient overexpression studies, DNA plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen). Stable overexpression and silencing were obtained by transducing IMR-90 and HEK 293T cells with retroviral or lentiviral vectors. The efficiency of knock-down or overexpression was controlled by Western blotting. Infected cells were selected with 2 μg/ml puromycin (Sigma-Aldrich).

Colony formation and SA-β-gal

For colony-formation or foci assay, early-passage MEFs (passage 5) cells were plated at low density into 60-mm cell culture plates. When sufficient colonies were visible, typically after 2-3 weeks, cells were washed twice in PBS before fixing in ice-cold 70% methanol for 30 min and then stained by 0.2% crystal violet for 0.5-1 h. The following day, cells were rinsed in PBS and air dried. For SA-β-Gal, MEFs were fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 10 min and stained for SA-β-Gal according to manufacturer's instructions (Cell Signaling) overnight at 37 °C.

Protein stabilization analysis

For protein stabilization analysis, HeLa cells were transfected with the indicated constructs. After transfection for 48 h, cells were treated with CHX (20 μg/ml). The cell lysates were prepared and analyzed by Western blot analysis. After CHX treatment, endogenous or exogenous Myc levels were quantified by densitometric scanning in the image J program.

Co-immunoprecipitation assays, immunoblotting, and antibodies

To study endogenous WSB1/ATM binding, the cells were treated with or without 10 μM MG132 for 2 h. One confluent 15-cm dish of HEK 293T cells was lysed by sonication in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl2, 1 mM EDTA, and 0.5% Nonidet P-40) containing 50 mM b-glycerophosphate,10 mM NaF, and 1 mg/ml each of pepstatin A and aprotinin, freshly supplemented with a protease inhibitor cocktail (Roche). Prior to immunoprecipitation, protein-A-bound agarose beads were incubated overnight with ATM (Abcam, ab32420), p-ATM (Abcam, ab81292), WSB1 (Abcam, ab68953; Sigma, HPA003293; Proteintech, 1166-1-AP), Ki-67 (Abcam, ab15580), p53 (DO-1; SantaCruz, sc-126), p16 (Abcam, ab51243), and ATR antibodies in PBS with 5% BSA at 4 °C. We used WSB1 antibody of ab68953 for most data (HPA003293 for IHC). We then added to the extracts before immunoprecipitation with protein-A sepharose at 4 °C for 4 h. After three washings in a binding buffer, co-purified proteins were analyzed by Western blotting. For the map** of the ATM-interacting domain of WSB1, HEK 293T cells were transfected with a 1 μg/10 cm dish of either wild-type or mutant forms of Myc-WSB1 and harvested in the presence of 10 μM MG132. Cell extract was then subjected to immunoprecipitation using anti-Myc. For removing heavy-chain, heavy- or light-chain-specific anti-mouse and anti-rabbit IgG, secondary antibodies were obtained from Jackson Immunoresearch. Anti-α-tubulin, Myc, and HA mouse antibodies were purchased from Sigma. To study WSB1 dimerization and/or monomerizaton, reduced samples were prepared with a reducing sample buffer containing β-ME and SDS, and then boiled. Non-reducing samples were put in the sample buffer without β-ME, which were not boiled. To study WSB1 phosphorylation by CDK2, we used an anti-Phospho-(Thr) MAPK/CDK substrate (Cell Signaling). Antibodies used in the other studies included the following: CDK1 (Abcam, ab32384), CDK2 (Abcam, ab64669), and CDK4 (Abcam, ab137675).

In vivo ubiquitination assays

For in vivo ubiquitination, including domain map** of WSB1, cells were transfected with ubiquitin-His plasmid together with Myc or Myc-WSB1, followed by treatment with MG 132 (10 μM). Forty-eight hours post-transfection, cells were lysed by Urea lysis buffer (8 M urea, 0.1 M Na2HPO4, 0.1 M Tris/HCl (pH 8.0), 0.05% Tween 20, and 0.01 M imidazole). After centrifugation, the supernatants were collected and incubated with 20 ml Ni-NTA agarose beads (Qiagen) for 4 h at 4 °C. The precipitates were washed three times with Urea wash buffer (8 M urea, 0.1 M Na2HPO4, 0.1 M Tris/HCl (pH 8.0), 0.05% Tween 20, and 0.02 M imidazole) and Native wash buffer (0.1 M Na2HPO4, 0.1 M Tris/HCl (pH 8.0), 0.05% Tween 20, and 0.02 M imidazole), boiled with SDS loading buffer, and then subjected to SDS-PAGE followed by immunoblot analysis.

In vitro binding assay

GST fusion proteins were prepared following standard protocol79. For in vitro biding assays, ATM (8 deleted mutants) GST fusion proteins bound to the GSH Sepharose were incubated with cell lysates. After washing, the bound proteins were separated by SDS-PAGE and immunoblotted with indicated antibodies.

Immunofluorescence

For immunofluorescence staining, MEF and IMR-90 cells were plated on glass cover slips and infected with the indicated constructs. Cells were then fixed in 3.7% paraformaldehyde for 10 min at room temperature and stained using standard protocols. Immunofluorescence images were taken using fluorescent microscopy (Nikon Microscope, Melville, NY, USA).

Proliferation and viability analysis

For proliferation staining, MEFs were plated on glass cover slips and infected with the indicated constructs. Cells were then fixed and stained with Ki-67 antibody. For viability staining, MEF cells were infected with the indicated constructs and were seeded in six-well plates and cultured for 15 days. Cells were then washed three times with PBS and stained with 0.2% crystal violet for 30 min before cell counting under microscopy.

In vitro kinase assay

HEK 293T cells were transfected with Flag-CDK2 and V5-CyclinE. Forty-eight hours later, the cells were collected and the cell lysates were subjected to immunoprecipitated anti-Flag antibody. The protein conjugated to the beads were eluted with Flag peptides and subjected to in vitro kinase assay, or the active CDK2/CyclinE protein from Millipore was used as kinase in the assay. GST and GST-WSB1 expressed in bacteria were purified and subjected to kinase assay as substrates. The phosphorylation of WSB1 by CDK2 was examined using an anti-CDK substrate antibody.

Reverse transcription-PCR of cDNA

RNA preparation and cDNA were described previously80. The following primers were used:

WSB1 forward 5′-TCTCCTGACTCTTCTATGCTGTGT-3′

reverse 5′-CATGGTGTATTTATCCATATTCCAAA-3′

ATM forward 5′-GTGTTCTGAAATTGTGAACCATGAGTCTAGT-3′

reverse 5′-TGGTATCTTCATTAAAAACCTGGTGACAGA-3′

forward 5′-GATGGAGAAAGTAGTGATGAGC-3′

reverse 5′-AGTCACCAGATTTCCATATTCTC-3′

The β-actin sequence was described previously80.

Electroporation

For complementation experiments, Vector, Flag-ATM-WT, or Flag-ATM mutant plasmids (K1109R, K1114R, K1323R, K1572R, K2025R, and 2KR), Flag-CDK1, Flag-CDK2, Flag-CDK4, or HA-WSB1 were electroporated into ATM KO MEFs using GenePulser Xcell (Bio-Rad). Pellet cells were resuspended in 10 ml of DMEM. The cells were either re-suspended (107) in 400 μl of transfection serum-free medium and 10-40 μg of plasmid DNA added or re-suspended to the cells (5 × 107) in 400 μl of transfection serum-free medium and 40 μg of plasmid DNA added. Electroporation used the following settings: square wave pulse, 200 V, 30 ms, and 4 mm cuvette. The cuvette was placed on ice for 5-10 min. Cells were transferred to a 60-mm dish and 4 ml of media added (15% serum; without antibiotics and selective agent). Cells were then incubated overnight and media was changed at the next day.

Mouse xenograft tumor model

For MEF xenograft experiments, cells were infected (or transfected) with the indicated constructs (by electroporation) and injected into nude mice. Equal numbers (1 × 106) of infected MEF cells expressing luciferase mixed at a 1:1 dilution with Matrigel (Collaborative Research) were implanted in the backs of athymic nude mice. For CDK-related xenograft experiments, mice were treated with CDKIs, such as RO-3306 (CDK1 inhibitor), SNS-032 (BMS-387032, CDK1 inhibitor) and PD0332991 (CDK4 inhibitor). Tumor growth was monitored using calipers and visualized with a bioluminescence-based IVIS system (Caliper LifeScience). Tumor growth was measured using a Vernier caliper at the indicated times after injection, and the tumor volume was calculated as length × width × height. Tumor size was monitored by measuring mice two times a week. When tumors reached 2 cm in diameter, mice were euthanized.

Immunohistochemistry

All of step for IHC were prepared following standard protocol. Briefly, immunohistochemical cytokeratin staining was performed on formalin-fixed, paraffin-embedded tissue using an indirect immunoperoxidase technique. Sections mounted on silanized slides were dewaxed in xylene, dehydrated in ethanol, boiled in 0.01 M citrate buffer (pH 6.0) for 20 min in a microwave oven and then incubated with 3 % hydrogen peroxide for 5 min. After washing with PBS, the slides were incubated in 10 % normal BSA for 5 min, followed by incubation for 45 min with monoclonal mouse anti-human WSB1 (1:200, Sigma-Aldrich) and anti-ATM (1:100, Abcam). After washing, sections were incubated with labeled polymer (Bond Polymer Refine Detection) and diaminobenzidine. The sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted.

Statistical analysis

Each assay was performed in triplicate and independently repeated at least three times. The results are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism software (version 4.02; GraphPad Software, San Diego, CA, USA). One-way analysis of variance followed by t-testing was used to compare the results. A difference was considered significant if P < 0.05. Statistical significance was defined as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Author Contributions

SBL, JJK, and ZL designed and performed most of the experiments, analyzed data, and prepared the manuscript as lead authors. SYY, SHK, SAH, JML, and SYT provided clinical samples and analyzed data. PY and BG performed animal experiments. ZL designed and supervised the study.

Competing Financial Interests

The authors declare no competing financial interests.