WIP1, a Homeostatic Regulator of the DNA Damage Response, Is ...

Molecular Cell

Article WIP1, a Homeostatic Regulator of the DNA Damage Response, Is Targeted by HIPK2 for Phosphorylation and Degradation Dong Wook Choi,1,9 Wooju Na,1,9 Mohammad Humayun Kabir,2 Eunbi Yi,3 Seonjeong Kwon,4 Jeonghun Yeom,2,5 Jang-Won Ahn,1 Hee-Hyun Choi,6 Youngha Lee,7 Kyoung Wan Seo,1 Min Kyoo Shin,1 Se-Ho Park,3 Hae Yong Yoo,7 Kyo-ichi Isono,8 Haruhiko Koseki,8 Seong-Tae Kim,6 Cheolju Lee,2,5 Yunhee Kim Kwon,4 and Cheol Yong Choi1,* 1Department

of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Republic of Korea 3School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea 4Department of Biology and Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea 5Department of Chemical Biology, University of Science and Technology, Yuseong-gu, Daejeon 305-350, Republic of Korea 6Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea 7Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710, Republic of Korea 8RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan 9These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.06.010 2BRI,

SUMMARY

WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKa2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATMmediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IRinduced g-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/ mice upon g-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR. INTRODUCTION Ataxia telangiectasia mutated (ATM) orchestrates proper double-strand break (DSB) repair and cell-cycle checkpoint activation to prevent cells with unrepaired DSBs (Lavin and Kozlov, 374 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc.

2007). ATM-mediated signal transduction is reversibly controlled by the type 2C phosphatase WIP1 (Shreeram et al., 2006a). WIP1 functions as a homeostatic regulator in DSB repair signaling, where WIP1 dephosphorylates numerous targets of the ATMdependent signaling pathway after completion of DSB repair to return cells to a prestress state (Lu et al., 2008). In concert with the homeostatic function of WIP1, the levels of WIP1 were gradually induced by p53 at the transcriptional level following exposure to ionizing radiation (IR) (Fiscella et al., 1997; Rossi et al., 2008). Genetic analysis indicates that the Wip1 deficiency suppresses oncogene-induced tumorigenesis (Bulavin et al., 2004; Shreeram et al., 2006b), suggesting that WIP1 is an oncogene and that its overexpression could contribute to tumor formation through inhibition of ATM and p53 functions. ATM is an apical protein kinase involved in the response to IR. Several protein kinases, including checkpoint kinase 1 (CHK1), CHK2, and AMP-activated protein kinase (AMPK), are phosphorylated following IR in an ATM-dependent manner (Matsuoka et al., 2007). AMPK is a heterotrimeric enzyme composed of a catalytic a subunit and noncatalytic b and g subunits (Viollet et al., 2010). In response to IR, activated AMPK induces the expression of p53 and p21 for cell-cycle arrest and G2/M checkpoint activation (Sanli et al., 2010). In addition, depletion of AMPK impairs clonogenic cell survival in response to IR (Zannella et al., 2011). However, the molecular target(s) of AMPK in response to IR remains to be elucidated. HIPK2 was initially identified as a corepressor for NK family homeoproteins (Kim et al., 1998). Subsequently, HIPK2 was found to participate in various cellular processes such as apoptosis, cell proliferation, and the DNA damage response (Calzado et al., 2007; Choi et al., 2008; Kim et al., 2009; Rinaldo et al., 2007; Sombroek and Hofmann, 2009). Several lines of evidence strongly indicate that HIPK2 functions as a tumor suppressor (D’Orazi et al., 2012). Notably, HIPK2 expression and

Molecular Cell Phosphorylation and Degradation of WIP1 by HIPK2

function was impaired in differentiated thyroid carcinomas as well as colorectal and breast cancers (D’Orazi et al., 2006; Lavra et al., 2011; Pierantoni et al., 2007). Loss of heterozygosity was also observed in thyroid carcinomas (Lavra et al., 2011) and mouse models (Mao et al., 2012). HIPK2 is known to be a downstream target of ATM in response to IR (Dauth et al., 2007), and it phosphorylates the Ser46 residue of p53 for induction of apoptosis. However, little is known about its role in the process of DSB repair. In this study, we identify HIPK2 as a protein kinase acting in WIP1 phosphorylation and degradation by screening prolinedirected protein kinases. In addition, we identify AMPKa2 as an upstream protein kinase that phosphorylates HIPK2 in response to IR. WIP1 is maintained at a low level by HIPK2mediated constitutive phosphorylation and degradation. To achieve homeostatic function, WIP1 levels increased after g-irradiation by dissociating from HIPK2 following phosphorylation by AMPKa2. Accordingly, HIPK2-mediated downregulation of WIP1 in the initiation of DSB signaling is crucial for the DNA repair process and checkpoint activation in response to IR. This study advances our understanding of how WIP1 functions as a homeostatic regulator in response to IR. RESULTS Phosphorylation-Mediated Proteasomal Degradation of WIP1 and Identification of HIPK2 for WIP1 Phosphorylation WIP1 protein is maintained at a low level in cells growing under normal conditions, and expression is induced by p53 after g-irradiation (Fiscella et al., 1997; Rossi et al., 2008). However, WIP1 levels increased following g-irradiation, independent of messenger RNA (mRNA) levels in human embryonic kidney (HEK) 293 cells (Fuku et al., 2007). We hypothesized that WIP1 might be regulated at the protein level as well as the transcriptional level as a fast and efficient way to maximize quick responses to DNA damage stress such as ionizing radiation (IR). To test this idea, WIP1 protein levels in response to IR were analyzed in the presence or absence of MG132 in both p53-positive (HCT116 p53+/+) and p53-negative cells (HCT116 p53 / ). WIP1 levels were elevated both before and after g-irradiation in the presence of MG132, even in p53-negative HCT116 cells (Figure 1A), suggesting that WIP1 undergoes proteasomal degradation constantly in cells growing under normal conditions. Since much of DNA damage signaling is relayed by phosphorylation, it is plausible that WIP1 degradation might be regulated by phosphorylation. To determine whether WIP1 phosphorylation is associated with its degradation, WIP1 levels were measured following the administration of cells with increasing amounts of the phosphatase inhibitor okadaic acid. The levels of both endogenous and overexpressed WIP1 decreased, and the migration of WIP1 on SDS-PAGE gels shifted upward in proportion to the amount of okadaic acid (Figure 1B). The downregulation of WIP1 levels was recovered by treating cells with MG132, confirming that phosphorylation of WIP1 results in proteasomal degradation (Figure 1C). The half-life of phosphorylated WIP1 was markedly shorter than that of WIP1 protein (Figure 1D), and WIP1 phosphorylation was detected in unstressed cells,

gradually diminished at the early stages of DNA damage response (DDR) (up to 7 hr), and was recovered 24 hr after IR (Figure 1E). These results indicate that WIP1 is constantly phosphorylated and subjected to proteasomal degradation, and the phosphorylation-mediated degradation is gradually inhibited in response to IR. In an attempt to identify the phosphorylation sites of WIP1 in unstressed cells, mass spectrometric analysis was performed with hemagglutinin (HA)-WIP1 immunoprecipitated with anti-HA antibody from HCT116 cell lysates expressing HA-WIP1. Mass analysis of WIP1 phosphopeptides indicated that Ser85 was highly phosphorylated (Figure S1 available online). Subsequently, a phospho-specific antibody against the WIP1 Ser85 residue was raised, and its specificity was verified (Figure S1). To identify the protein kinase(s) responsible for WIP1 phosphorylation and potential degradation, we screened proline-directed protein kinases for WIP1 phosphorylation at the Ser85 residue with immunoblotting using pSer85-specific anti-WIP1 antibody, since Ser85 was immediately preceded by a proline. Among 29 proline-directed protein kinases, including the cyclin-dependent kinase (CDK) family, mitogen-activated protein kinase (MAPK) family, dual-specificity tyrosine phosphorylation-regulated kinase (DYRK)/HIPK family, and other proline-directed protein kinases, HIPK2 was found to highly phosphorylate WIP1 at the Ser85 residue (Figures 1F and S2). To test whether HIPK2 is a negative regulator of WIP1 under physiological conditions, we determined the effects of either WIP1 or HIPK2 knockdown on the phosphorylation of DSB signaling regulators. As demonstrated previously, WIP1 depletion resulted in hyperphosphorylation of ATM, CHK2, and H2AX in response to IR, whereas HIPK2 depletion led to WIP1 stabilization and consequent dephosphorylation of its targets such as ATM, CHK2, and H2AX (Figure 1G). HIPK2 depletion did not affect WIP1 mRNA levels (Figure 1H). These results suggest that HIPK2 is a protein kinase that may induce phosphorylation and proteasomal degradation of WIP1. HIPK2 Mediates Ubiquitination and Proteasomal Degradation of WIP1 To further confirm the role of HIPK2 on WIP1 stability, the dynamics of endogenous WIP1 levels before and after g-irradiation were determined in both normal and HIPK2 knockdown HCT116 cells. Endogenous WIP1 levels were low in unstressed cells, gradually rose upon g-irradiation, and peaked at around 7 hr after irradiation. Upon HIPK2 knockdown by small interfering RNA (siRNA), WIP1 levels were significantly higher in both unstressed cells and g-irradiated cells, which were restored by expression of siRNA-resistant HIPK2 (Figure 2A). The same results were also observed in hipk2 knockout (KO) mouse embryonic fibroblasts (MEFs) (Figure 2B). In addition, expression of increasing amounts of HIPK2 resulted in WIP1 degradation in a dose-dependent manner. The decrease in WIP1 levels in the presence of HIPK2 was blocked by administration of the proteasome inhibitor MG132 (Figure 2C). Notably, the catalytic activity of HIPK2 was required for WIP1 degradation, since a catalytically inactive HIPK2 mutant could not induce WIP1 degradation (Figure 2D). To confirm whether HIPK2 causes a decrease in WIP1 levels, WIP1 stability was determined in the presence of cycloheximide. Overexpression of HIPK2 resulted in a markedly Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc. 375


Figure 1. HIPK2 Is a Protein Kinase that Phosphorylates WIP1 (A) HCT116 (p53+/+) and HCT116 (p53 / ) cells were exposed to g-irradiation in the presence or absence of MG132, and cells were analyzed for WIP1 and p53 levels at the indicated time points after g-irradiation. WIP1/actin ratios are depicted on the graph to the right. (B) HCT116 cells or HCT116 cells expressing HA-WIP1 were treated with increasing amounts of okadaic acid, a Ser/Thr phosphatase inhibitor, and endogenous WIP1 and HA-WIP1 levels were determined by immunoblotting. (C) HCT116 cells were treated with okadaic acid alone or in combination with MG132, and endogenous WIP1 levels were determined by immunoblotting. (D) HCT116 cells expressing HA-WIP1 were harvested at the indicated time points after cycloheximide (CHX) treatment, and lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-phospho-Ser antibody and anti-WIP1 antibody. (E) HCT116 cells expressing HA-WIP1 were harvested at the indicated time points after g-irradiation, and lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies. (F) HCT116 cells were transfected with Myc-WIP1 and expression plasmids encoding the indicated protein kinases. Lysates were immunoblotted with anti-Myc and anti-WIP1 (phospho-Ser85) antibody. The relative phosphorylation levels of WIP1 at the Ser85 residue were determined by phospho-WIP1/WIP1 ratios. (G) HCT116 cells were transfected with the indicated siRNAs and exposed to g-irradiation. Cell lysates were immunoblotted with the indicated antibodies. (H) WIP1 mRNA level was measured by quantitative real-time PCR in control or HIPK2-depleted cells. Data are represented as mean ± SEM. See also Figures S1 and S2.

reduced half-life of HA-WIP1 (Figure 2E). Depletion of HIPK2 using siRNA led to increased WIP1 stability (Figure 2F). Next, we determined whether WIP1 is modified by polyubiquitination in a HIPK2-dependent manner. Immunoblot analysis showed that polyubiquitination of WIP1 increased upon coexpression with wild-type HIPK2 in the presence of MG132, but the catalytically inactive HIPK2 mutant inhibited polyubiquitination of WIP1 in a dominant-negative fashion (Figure 2G). We also observed that HA-WIP1 is polyubiquitinated in wild-type MEFs and that this ubiquitination is inhibited by HIPK2 knockdown (Figure 2H, lane 3). When Myc-HIPK2 was reintroduced into hipk2 KO MEFs, the polyubiquitination of WIP1 was restored, showing 376 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc.

that HIPK2 destabilizes WIP1 protein and verifying that polyubiquitination of WIP1 depends on HIPK2 (Figure 2H, lane 6). Collectively, these results demonstrate that HIPK2 regulates the stability of WIP1 by inducing polyubiquitination and proteasome-dependent degradation of WIP1. HIPK2 Physically Interacts with and Phosphorylates WIP1 for Proteasomal Degradation As shown in Figures 2D and 2G, the catalytic activity of HIPK2 is required for the polyubiquitination and degradation of WIP1. Therefore, we first determined whether HIPK2 physically interacts with WIP1, which is prerequisite for WIP1 phosphorylation.


Figure 2. WIP1 Is Subjected to Proteasomal Degradation in a HIPK2 Catalytic Activity-Dependent Manner (A) HCT116 cells were transfected with control siRNA, siHIPK2, or siHIPK2 with siHIPK2-resistant Myc-HIPK2 expression plasmid and harvested at the indicated time points after g-irradiation. Cell lysates were analyzed by immunoblotting with anti-WIP1 and anti-HIPK2 antibodies. (B) Wild-type (WT) MEFs and HIPK2 null MEFs were harvested at the indicated time points after g-irradiation, followed by immunoblotting with anti-WIP1 and antiHIPK2 antibodies. (C) HCT116 cells were transfected with the HA-WIP1 expression plasmid alone or in conjunction with increasing amounts of Myc-HIPK2 expression plasmid. The levels of Myc-HIPK2 and HA-WIP1 were analyzed by immunoblotting with anti-Myc and anti-HA antibody, respectively. Transfection efficiencies were monitored by GFP expression. (D) HEK 293T cells were transfected with HA-WIP1 expression plasmid alone or in combination with increasing amounts of WT or catalytically inactive Myc-HIPK2 expression plasmid (KD). The levels of Myc-HIPK2 and HA-WIP1 were analyzed by immunoblotting with anti-Myc and anti-HA antibody, respectively. (E) HeLa cells expressing HA-WIP1 alone or HA-WIP1 with Myc-HIPK2 were harvested at the indicated time points after CHX treatment, and cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. The relative levels of HA-WIP1 in the presence or absence of HIPK2 are shown on the graph to the right. (F) HCT116 cells were transfected with control siRNA or siRNA targeting HIPK2 and were harvested at the indicated time points after cycloheximide treatment. Cell lysates were analyzed by immunoblotting with anti-WIP1 and anti-HIPK2 antibodies. The relative levels of endogenous WIP1 in either wild-type (siCon) or HIPK2-depleted cells (siHIPK2) are shown on the graph. (G) HCT116 cells were transfected with the indicated plasmids. Cells were treated with MG132 3 hr prior to harvest. Cell lysates were applied to Ni-NTA columns, and both the eluate and cell lysates were analyzed by immunoblotting with the indicated antibodies. (H) Wild-type MEFs and HIPK2 null MEFs were transfected with expression plasmids and/or siRNA targeting HIPK2 as indicated and treated with MG132 4 hr prior to harvest. Cell lysates were immunoblotted with the indicated antibodies or immunoprecipitated with anti-HA antibody, followed by immunoblotting with antiubiquitin (Ub) antibody.

In both a coimmunoprecipitation assay and glutathione S-transferase (GST) pull-down assay, HIPK2 interacted with WIP1 in vivo and in vitro, respectively (Figure S3). Since HIPK2 was identified as the protein kinase that phosphorylates WIP1 at the Ser85 residue (Figure 1F) and found to physically interact with WIP1, we explored the effects of WIP1 phosphorylation by HIPK2 on WIP1 stability. Affinity-purified GST-WIP1 was strongly phosphorylated by GST-HIPK2 in the presence of g-32P-labeled ATP (Figure 3A). Mass spectrometric analysis was carried out

using in vitro phosphorylated GST-WIP1 to identify phosphorylation sites (Kabir et al., 2012). Analysis of the phosphoamino acids of WIP1 revealed that both Ser85 and Ser54 are phosphorylation sites, confirming that HIPK2 is a protein kinase for WIP1 phosphorylation at Ser54 as well as Ser85 (Figure S4). Consistently, domain analysis of WIP1 indicated that full-length and aminoterminal HA-WIP1 (amino acids [aa] 1–100), which contains both phosphorylation sites, but not middle (aa 101–372) or carboxy-terminal WIP1 (aa 373–605), were degraded by HIPK2 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc. 377


Figure 3. WIP1 Is Phosphorylated by HIPK2 at the Ser54 and Ser85 Residues In Vitro and In Vivo (A) Affinity-purified GST or GST-WIP1 protein was incubated with affinity-purified GST-HIPK2 (1–629) in the presence of g-32P-labeled ATP. CBB indicates the affinity-purified GST and GST-WIP1 used in the in vitro kinase assay. (B) Wild-type HA-WIP1 and deletion mutants were transfected into HCT116 cells in combination with increasing amounts of Myc-HIPK2 expression plasmids. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Transfection efficiencies were monitored by GFP expression. (C) Affinity-purified wild-type GST-WIP1 (1–100) and its mutants were subjected to an in vitro kinase assay with GST-HIPK2 (1–629) in the presence of g-32Plabeled ATP. (D) Wild-type HA-WIP1 or serine substitution mutants were transfected into HeLa cells with or without the Myc-HIPK2 expression plasmid. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (E) Wild-type HA-WIP1 or the HA-WIP1 Ser54/85Ala mutant was transfected into HeLa cells in combination with the Myc-HIPK2 expression plasmid. Cells were treated with CHX and harvested at the indicated time points, followed by immunoblotting with anti-HA and anti-Myc antibodies. The relative stabilities of HA-WIP1 and the phosphorylation-defective mutant are plotted on the graph to the right. (F) HCT116 cells were transfected with Myc-HIPK2 alone or in combination with wild-type HA-WIP1 or HA-WIP1 serine substitution mutants. The transfected cells were treated with MG132 4 hr prior to harvest, followed by immunoprecipitation with anti-HA antibody. Cell lysates and the immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (G) HeLa cells were transfected with the HA-WIP1 expression plasmid alone or in combination with the WT or catalytically inactive Myc-HIPK2 expression plasmid (KD). Cells were treated with MG132 3 hr prior to harvest, as indicated. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (H) HIPK2 null MEFs were transfected with wild-type Myc-HIPK2 or the catalytically inactive Myc-HIPK2 expression plasmid. The transfected null MEFs were harvested, and lysates were analyzed by immunoblotting with the indicated antibodies. The relative levels of endogenous WIP1 are indicated by the WIP1/actin ratios at the bottom of the panel. See also Figures S3 and S4.

coexpression (Figure 3B). To further analyze WIP1 phosphorylation by HIPK2, Ser54 and Ser85 were substituted with alanine residues, either singly or in combination. In an in vitro phosphorylation assay carried out in the presence of g-32P-labeled ATP, single mutations (Ser54Ala or Ser85Ala) reduced phosphorylation, and the double mutant (Ser54/85Ala) showed a marked reduction in phosphorylation to almost negligible levels (Figure 3C). The catalytic activity of the WIP1 double mutant against phosphorylated CHK2 was not altered compared to wild-type WIP1 (Figure S2F). To determine whether WIP1 phosphorylation is associated with degradation, the stabilities of WIP1 mutants 378 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc.

were examined in the presence of HIPK2. The WIP1 Ser54Ala or Ser85Ala single mutant was degraded to almost the same degree as wild-type WIP1, whereas the WIP1 Ser54/85Ala double mutant was resistant to HIPK2-mediated degradation (Figure 3D). These results were verified again by examination of half-life and polyubiquitination of WIP1 phosphorylation-defective mutants. The half-life of the HA-WIP1 Ser54/85Ala mutant was markedly extended compared to wild-type HA-WIP1 in the presence of HIPK2 expression (Figure 3E). Polyubiquitination of the HA-WIP1 Ser54/85Ala mutant was accordingly reduced, whereas the wild-type and single mutants of WIP1 showed


efficient polyubiquitination by HIPK2 (Figure 3F). These results demonstrate that WIP1 phosphorylation at either the Ser54 or Ser85 residue is required for its proteasomal degradation. The relationship between WIP1 phosphorylation by HIPK2 and its degradation was further analyzed by immunoblotting using a pSer85-specific anti-WIP1 antibody. Coexpression of the HIPK2 catalytically inactive mutant did not induce WIP1 degradation and inhibited WIP1 phosphorylation in a dominant-negative manner (Figure 3G, lane 5). Strong WIP1 phosphorylation at the Ser85 residue was detected only when wild-type HIPK2 was coexpressed with WIP1 and WIP1 degradation was blocked via MG132 treatment (Figure 3G, lane 4). We also found that WIP1 degradation depends on the catalytic activity of HIPK2 in MEFs. When either wild-type Myc-HIPK2 or the kinase-dead Myc-HIPK2 mutant was reintroduced into hipk2 KO MEFs, endogenous WIP1 degradation was restored by wild-type HIPK2, not by the kinase-dead HIPK2 mutant (Figure 3H), demonstrating that WIP1 phosphorylation by HIPK2 plays a causative role in WIP1 destabilization. Ionizing Radiation Prevents Phosphorylation of WIP1 with HIPK2 Given that WIP1 levels should be elevated to terminate the ATMmediated signaling cascade following g-irradiation, WIP1 phosphorylation should be concordantly regulated. We addressed the dynamics of WIP1 phosphorylation during the cellular response to IR. Immunoblotting using anti-pSer85 WIP1 antibody indicated that WIP1 was phosphorylated before irradiation and that the phosphorylation was gradually reduced following g-irradiation. Notably, the phosphorylation of WIP1 was conversely correlated with WIP1 protein levels and was not detected in HIPK2-depleted cells (Figure 4A). Consistent with this observation, polyubiquitination of WIP1 occurred in unstressed cells, but not in HIPK2-depleted cells, and the intensity of polyubiquitinated WIP1 gradually decreased following g-irradiation (Figure 4B). Accordingly, WIP1 stability was increased after g-irradiation (Figure 4C). Taken together, these results demonstrate that phosphorylated WIP1 is constitutively degraded in unstressed cells and that phosphorylation-mediated degradation of WIP1 is gradually prevented by IR. Since interaction of HIPK2 with its binding partners was inhibited after DNA damage (Choi et al., 2008; Winter et al., 2008), we determined whether dissociation of WIP1 from HIPK2 is the reason for reduction of WIP1 phosphorylation after IR. Coimmunoprecipitation assays indicated that endogenous WIP1 was associated with HIPK2 in unstressed cells, and this interaction was reduced at around 2 hr after g-irradiation and gradually restored within 24 hr after g-irradiation (Figures 4D and S5A), consistent with the results showing no phosphorylation 2 hr after g-irradiation (Figure 4A). The dissociation of HIPK2 from WIP1 in response to IR was attributed to HIPK2 phosphorylation, since phosphorylation of HIPK2 following IR affects HIPK2 interaction with WIP1 as shown in a GST pull-down analysis (Figure S5B, lane 2). Interestingly, in ATM-depleted cells, WIP1 remained associated with HIPK2, and WIP1 phosphorylation was not reduced in response to IR, but this was not the case in ataxia telangiectasia and Rad3-related protein (ATR)-depleted cells (Figures 4E, 4F, and S5C). These results indicate that ATM regulates HIPK2-WIP1 interaction and

WIP1 phosphorylation by HIPK2. Since we could not observe direct phosphorylation of HIPK2 by ATM in vitro or in vivo, mass spectrometric analysis was performed with HA-HIPK2 immunoprecipitated with anti-HA antibody from the lysates of g-irradiated HCT116 cells expressing HA-HIPK2. We found that Thr112 and Ser114, potential AMPK target sequences, are major phosphorylation sites of HIPK2 following g-irradiation (Figure S6). To determine whether HIPK2-mediated WIP1 phosphorylation following IR is affected by AMPK depletion, WIP1 phosphorylation was analyzed in the presence or absence of AMPK. Immunoblotting indicated that WIP1 phosphorylation by HIPK2 was not reduced following IR in cells depleted of ATM or AMPKa2, but not AMPKa1 (Figure 4G). Accordingly, WIP1 stability was reduced by AMPKa2 depletion following IR (Figure 4H), and WIP1 was stabilized by overexpression of wild-type AMPKa2, but not by catalytically inactive AMPKa2 (Figure S5D). Furthermore, AMPKa2 activation was induced by ATM (Figure S5E) (Sanli et al., 2010; Zannella et al., 2011), and WIP1 stabilization by AMPKa2 coexpression was reversed by ATM depletion following IR (Figure S5F). These results demonstrate that ATM stabilizes WIP1 indirectly via the catalytic activity of AMPKa2 in response to IR. Next, a series of experiments were performed to determine whether HIPK2 is phosphorylated by AMPKa2. Immunoblotting indicated that HIPK2 associated with AMPKa2 in HCT116 cells (Figure S5G), and HIPK2 was strongly phosphorylated by AMPKa2 at the N and C termini in vitro (Figure S5H). Immunoblotting using phospho-AMPK substrate antibody (recognizing LXRXXpS/pT) indicated that HIPK2 phosphorylation by AMPKa2 was strongly induced upon g-irradiation (Figures 4I and S5I). These results indicate that HIPK2 is a substrate of AMPKa2 in vitro and in vivo. Notably, time course experiments demonstrated that ATM-dependent AMPK phosphorylation was followed by AMPK-mediated HIPK2 phosphorylation, which resulted in WIP1 stabilization and a decrease of WIP1 phosphorylation (Figure 4J). Potential HIPK2 phosphorylation sites were deduced by searching for the AMPK consensus sequence and from the results of mass spectrometry (Figure S6). Site-directed mutagenesis of Thr112 and Ser114 in the N terminus, and Thr1107 in the C terminus markedly reduced HIPK2 phosphorylation by AMPKa2 in vitro (Figure S5J). These sites match well to the consensus sequence of AMPK phosphorylation targets (Figure S5J). Wild-type HIPK2 phosphorylation was induced upon g-irradiation, but the HIPK2 3A mutant (Thr112Ala, Ser114Ala, and Thr1107Ala) was not phosphorylated by AMPK (Figure 4K), showing that HIPK2 phosphorylation by AMPKa2 in vitro is recapitulated in vivo in response to g-irradiation. To determine whether HIPK2 phosphorylation by AMPKa2 can be attributed to the inhibition of WIP1 phosphorylation-mediated degradation, we determined whether WIP1 stability is affected by expression of the HIPK2 3A mutant. A coimmunoprecipitation assay indicated that wild-type HIPK2-WIP1 interaction was reduced upon g-irradiation, while interaction of the HIPK2 3A mutant with WIP1 was not affected by g-irradiation (Figure 4L). Accordingly, WIP1 stability was markedly reduced upon coexpression of the HIPK2 3A mutant, compared to coexpression of wild-type HIPK2 (Figure 4M). Notably, WIP1 phosphorylation was inhibited following IR in mock-depleted cells (Figure 4N, lanes 1 and 2) Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc. 379


Figure 4. Ionizing Radiation Inhibits Phosphorylation of WIP1 by HIPK2 (A) HCT116 cells were transfected with control siRNA or siRNA targeting HIPK2. Half of the cells expressing control siRNA were treated with MG132 4 hr prior to harvest. Endogenous WIP1 levels and the extent of phosphorylation were measured by immunoblotting with anti-WIP1 and anti-pSer85-WIP1 antibody, respectively. (B) HCT116 cells were transfected with control siRNA or siRNA targeting HIPK2 in combination with the HA-WIP1 expression plasmid, followed by treatment with MG132. Cells were harvested at the indicated time points after g-irradiation, and lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies. (C) HCT116 cells were either untreated or g-irradiated, and they were treated with CHX followed by harvest at the indicated time points. Cell lysates were analyzed by immunoblotting using anti-WIP1 antibody. The relative stability of endogenous WIP1 is plotted on the graph. (D) HCT116 cells were left untreated or exposed to g-irradiation. Cells were harvested at the indicated time points, and lysates were immunoprecipitated with antiHIPK2 antibody, followed by immunoblotting with the indicated antibodies. (E) H1299 cells were transfected with control siRNA, siATM, or siATR in combination with expression plasmids encoding Myc-HIPK2 and HA-WIP1, and cells were left untreated or g-irradiated. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with the indicated antibodies. (F) H1299 cells were transfected with control siRNA, siHIPK2, and siATM, and cells were left untreated or g-irradiated. Cells were treated with MG132 4 hr prior to harvest. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (G) H1299 cells were transfected with control siRNA, siATM, siAMPKa1, or two independent siAMPKa2 (#1 or #2), and cells were analyzed with the same procedure as in (F). (H) H1299 cells were transfected with control siRNA or siAMPKa2 followed by g-irradiation, and they were harvested at the indicated time points after CHX treatment. Cell lysates were analyzed by immunoblotting with anti-WIP1 and anti-AMPKa2 antibodies. (I) H1299 cells were transfected with control siRNA, siAMPKa1, or siAMPKa2 in combination with expression plasmids encoding HA-HIPK2, and cells were left untreated or g-irradiated. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies.

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Figure 5. HIPK2 Depletion Impairs DNA Double-Strand Break Signaling in Response to Ionizing Radiation (A) HCT116 cells were transfected with control siRNA or siRNA targeting HIPK2, exposed to g-irradiation, and treated with wortmannin 3 hr prior to fixation of cells for immunostaining with anti-g-H2AX antibody. HIPK2 depletion by siRNA was confirmed by immunoblotting with anti-HIPK2 antibody. The number of g-H2AX foci was counted in an arbitrary area and is shown on the graph to the right. Data are represented as mean ± SEM. (B) HCT116 cells were transfected with control siRNA or one of two independent siRNAs targeting HIPK2 and then exposed to g-irradiation. Cells were harvested at the indicated time points, and lysates were immunoblotted with the indicated antibodies. (C) Wild-type MEFs and HIPK2 null MEFs were exposed to g-irradiation. Cells were harvested at the indicated time points, and lysates were immunoblotted with the indicated antibodies. (D) HCT116 cells were transfected with control siRNA, siHIPK2 alone, or both siHIPK2 and siWIP1. Cells were exposed to g-irradiation and harvested at the indicated time points. Cell lysates were immunoblotted with the indicated antibodies.

or HIPK2-depleted cells reconstituted with siHIPK2-resistant wild-type HIPK2 (lanes 5 and 6), but not in cells reconstituted with the HIPK2 3A mutant defective in phosphorylation by AMPKa2 (lanes 7 and 8). Collectively, WIP1 phosphorylation by HIPK2 was inhibited by g-irradiation via AMPKa2-mediated regulation of the HIPK2-WIP1 interaction, consequently resulting in stabilization of WIP1 by escape from phosphorylation-mediated proteasomal degradation.

HIPK2 Depletion Impairs DNA Repair Signaling in Response to Ionizing Radiation Since WIP1 is a homeostatic regulator for DNA repair signaling in response to IR, we explored the role of HIPK2 as an upstream regulator of WIP1 phosphatase in DSB repair signaling. Depletion of HIPK2 using siRNA resulted in a comparable decrease in the number of g-H2AX foci after g-irradiation (Figure 5A). Immunoblotting showed that phosphorylation of ATM, CHK2,

(J) HCT116 cells were transfected with wild-type HA-HIPK2, and cells were left untreated or exposed to g-irradiation. Cells were harvested at the indicated time points, and lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies. (K) H1299 cells were transfected with wild-type HA-HIPK2 or the HA-HIPK2 3A mutant (Thr112Ala, Ser114Ala, and Thr1107Ala), and cells were left untreated or irradiated. Immunoprecipitated HA-HIPK2 was subjected to immunoblotting with anti-HA and anti-phospho-AMPK substrate antibodies. (L) H1299 cells were transfected with wild-type Myc-HIPK2 or the Myc-HIPK2 3A mutant along with the HA-WIP1 expression plasmid, and lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with the indicated antibodies. (M) HeLa cells were transfected with wild-type Myc-HIPK2 or the Myc-HIPK2 3A mutant along with the HA-WIP1 expression plasmid. Following g-irradiation, cells were harvested at the indicated time points after CHX treatment. Cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (N) H1299 cells were transfected with siCon or siHIPK2. HIPK2-depleted cells were reconstituted with expression of siRNA-resistant wild-type HA-HIPK2 or the HA-HIPK2 3A mutant. Both siRNA-transfected and reconstituted cells were left untreated or g-irradiated. Cells were treated with MG132 4 hr prior to harvest. Cell lysates were analyzed by immunoblotting with anti-phospho-WIP1 and the indicated antibodies. See also Figures S5 and S6.

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and H2AX was markedly reduced in both HIPK2 knockdown HCT116 cells (Figure 5B) and hipk2 KO MEFs after g-irradiation (Figure 5C). These results indicate that HIPK2 function is crucial in DSB signaling in response to IR. To confirm whether the effect of HIPK2 depletion on DSB signaling is mediated by WIP1 degradation, the activation of key players upon g-irradiation was determined in either HIPK2-depleted or HIPK2/WIP1double-depleted HCT116 cells. As demonstrated above, HIPK2 depletion using siRNA resulted in WIP1 stabilization and dephosphorylation of WIP1 targets, whereas double depletion of HIPK2 and WIP1 restored the phosphorylation of WIP1 targets such as ATM, H2AX, or CHK2 (Figure 5D). Taken together, these results strongly suggest that HIPK2-mediated downregulation of WIP1 is crucial in the DSB repair signaling pathway. HIPK2 Heterozygous Mice Are Highly Susceptible to Ionizing Radiation-Induced Death Next, we determined the effects of HIPK2 depletion on the cellular response to IR. The impairment of CHK1 phosphorylation upon HIPK2 depletion (Figure 1G) led us to analyze G2/M checkpoint activation. In normal cells, phospho-histone H3 levels were decreased 5-fold upon g-irradiation (Xu et al., 2002), whereas histone H3 phosphorylations were reduced 2.5-fold in HIPK2-depleted cells, suggesting that activation of the G2/M checkpoint is inhibited by HIPK2 knockdown (Figure 6A). In addition, entry into S phase, as measured by bromodeoxyuridine (BrdU) incorporation, indicated that activation of the G1/S checkpoint was inhibited in HIPK2-depleted cells in response to g-irradiation (Figure 6B). To demonstrate further, cell survival was determined by observation of colony formation with increasing doses of g-irradiation. The depletion of HIPK2 markedly reduced survival rate in an irradiation dose-dependent manner, whereas the depletion of both HIPK2 and WIP1 restored survival rate to the level of control cells (Figure 6C). These results indicate that the effects of HIPK2 on DSB signaling are mediated via downregulation of WIP1 protein levels. Moreover, survival rates were determined with HIPK2-depleted cells and WIP1depleted cells reconstituted with either siWIP1-resistant wildtype WIP1 or phosphorylation-defective WIP1 mutant. The survival rate of the reconstituted HCT116 cells expressing HAWIP1 was almost the same as that of normal cells, whereas expression of the HA-WIP1 Ser54/85Ala mutant reduced survival rate to almost the same level as that of HIPK2-depleted cells (Figure 6D). This result indicates that precise control of WIP1 levels by HIPK2-mediated phosphorylation and proteasomal degradation is crucial to the cellular signaling cascade in response to IR. Next, we determined whether HIPK2 function is crucial in DNA damage-induced repair signaling in the mouse model. Since HIPK2 is known to be haploinsufficient (Mao et al., 2012; Wei et al., 2007), hipk2 heterozygous mice were exposed to whole-body g-irradiation, and survival rates were determined. Immunoblotting of tissue extract from mouse ear punches showed that endogenous Wip1 is elevated, and H2AX phosphorylation following g-irradiation is impaired in hipk2+/ mice (Figure 6E). Notably, the survival rate of hipk2+/ was markedly reduced upon single whole-body g-irradiation as compared to wild-type mice (Figure 6F). These findings strongly illustrate that HIPK2 is a key regulator of DSB repair signaling and that 382 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc.

HIPK2-mediated downregulation of the WIP1 homeostatic regulator is crucial in repair signaling in response to IR. DISCUSSION WIP1 is a homeostatic regulator of the DSB signaling pathway and is known to be induced by p53 and other transcription factors at the transcriptional level after exposure to IR (Lowe et al., 2012). In addition, WIP1 transcripts were targeted by microRNA (miR)-16 to prevent premature termination of DSB repair signaling (Zhang et al., 2010). Here we show that another level of regulation exists to achieve full homeostatic function of WIP1 in the DSB signaling pathway. WIP1 is constitutively phosphorylated by HIPK2 (Figure 3), and phosphorylated WIP1 is subjected to proteasomal degradation in unstressed cells (Figures 2 and 7A). WIP1 phosphorylation and degradation continued to an early stage of DDR, and WIP1 was gradually stabilized by dissociation from HIPK2 after irradiation (Figures 4 and 7A). This finding adds another layer of complexity to the regulation of WIP1 levels after DNA damage. In the absence of HIPK2, a negative regulator of WIP1, WIP1 levels were elevated under normal conditions. In this cellular context, the ATM-driven DSB signaling cascade could not be properly activated because of the high levels of WIP1 (Figure 7B). Therefore, HIPK2-mediated downregulation of WIP1 levels allows ATM-mediated DSB signaling to create g-H2AX foci and activate the cell-cycle checkpoint, which is crucial to the initiation of the DSB repair signaling pathway. To achieve the homeostatic role of WIP1 after g-irradiation, WIP1 levels need to be upregulated. In the early stages of DDR, WIP1 production is inhibited by miR-16, and WIP1 protein is still phosphorylated by HIPK2 and subjected to proteasomal degradation. At the late stages, WIP1 protein is synthesized de novo by p53-mediated transcription induction, and the synthesized WIP1 is stabilized by escape from HIPK2mediated proteasomal degradation in order to achieve efficient upregulation of WIP1 protein levels (Figure 7A). WIP1 regulation by HIPK2 at the protein level is independent of p53 (Figure S7). In conclusion, WIP1 expression is finely regulated by coordination of transcriptional induction, targeting by microRNA, and proteasomal degradation. Signaling pathways need to be turned on or off in response to relevant cues. HIPK2-mediated WIP1 phosphorylation and proteasomal degradation were gradually terminated in an ATM-dependent manner following IR. Unexpectedly, AMPKa2 was identified as the upstream protein kinase that phosphorylates HIPK2. AMPKa2-mediated HIPK2 phosphorylation is critical for WIP1 to dissociate from HIPK2 (Figure 4). Taking together all of the events, we propose a model of an autoinhibitory loop for ATM regulation in which ATM activates AMPKa2 following IR, and activated AMPKa2 phosphorylates HIPK2 at multiple sites, resulting in WIP1 stabilization by escape from phosphorylated HIPK2 (Figure 7C). Since WIP1 is a negative regulator of ATM, this represents a mode of negative feedback regulation of ATM. Given that activated AMPK inhibits the mammalian target of rapamycin (mTOR) pathway to restrict catabolic energy consumption, AMPK may participate in both DSB repair homeostasis through WIP1 regulation and cellular energy homeostasis through mTOR regulation in response to


Figure 6. HIPK2 Heterozygous Mice Are Susceptible to IR-Induced Death (A) HCT116 cells were transfected with siCon or siHIPK2, and mitotic indexes before or after g-irradiation (2 Gy) were determined by phospho-histone H3 (pH3)/ propidium iodide (PI) fluorescence-activated cell sorting (FACS) analysis. Fold reduction of pH3-positive cells is shown on the graph to the right. Data are represented as mean ± SEM. (B) HCT116 cells were transfected with siCon or siHIPK2, and the incorporation of BrdU after g-irradiation (16 Gy) was measured by FACS analysis. Fold reduction of BrdU incorporation following g-irradiation is shown on the graph to the right. Data are represented as mean ± SEM. (C and D) HCT116 cells were transfected with siCon, siHIPK2, siWIP1, or both siHIPK2 and siWIP1 (C). HCT116 cells were transfected with siCon, siHIPK2, or siWIP1. WIP1-depleted cells were reconstituted with expression of siRNA-resistant wild-type WIP1 or the WIP1 Ser54/85Ala (2SA) mutant (D). The relative survival rates were calculated by counting colonies 7 days after g-irradiation, as shown on the graph. Data are represented as mean ± SEM. (E) Ear punches were taken from a wild-type or hipk2+/ mouse before or after 6 Gy g-irradiation. The tissue extracts were analyzed by immunoblotting with antiHIPK2, anti-WIP1, and anti-g-H2AX antibodies. (F) Wild-type and hipk2 heterozygous mice were whole-body irradiated (6 Gy), and the survival curves are shown.

g-irradiation. After completion of DSB repair, AMPK may be returned to a normal status, since ATM and its phosphorylation targets are inactivated by stabilized WIP1. Thus, it appears that AMPK involvement in an ATM-negative feedback loop provides a sensory node with which to coordinate the regulation of genome integrity and cellular energy homeostasis. Although AMPK was reported to induce cell-cycle arrest and G2/M checkpoint activation in response to IR (Sanli et al., 2010; Zan-

nella et al., 2011), further elucidation of AMPK targets following IR at the molecular level may uncover a point of crosstalk between DNA damage repair and cellular energy homeostasis. ATM and AMPK are activated in a variety of stress conditions in addition to ionizing radiation (Ditch and Paull, 2012; Mihaylova and Shaw, 2011). Therefore, it should be interesting to address whether HIPK2 is regulated by ATM and AMPK under different stress conditions. Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc. 383


Figure 7. Schematic Summary of HIPK2Mediated WIP1 Regulation in Unstressed and g-Irradiated Cells (A) Molecular diagram for HIPK2-mediated WIP1 phosphorylation and proteasomal degradation and AMPKa2-mediated inhibition of WIP1 phosphorylation by HIPK2. (B) Dynamics of relative WIP1 levels before or after g-irradiation in the presence or absence of HIPK2. The threshold denotes the level of WIP1 sufficient for initiation of DSB signaling termination. The DNA damage response is illustrated in two parts, the early and late phase, which occur before or after initiation of DSB signaling termination. (C) A schematic representation of the autoinhibitory loop of ATM following ionizing radiation. ATM phosphorylates AMPKa2 to induce inhibitory phosphorylation of HIPK2. In turn, WIP1 is stabilized by dissociation from HIPK2 and initiates termination of DDR by targeting ATM itself and ATM phosphorylation targets.

EXPERIMENTAL PROCEDURES Antibodies and Other Materials Rabbit anti-phospho-Chk1 (pS345, #2341), phospho-Chk2 (pT68, #2661), phospho-serine, ATM (#2873), H2AX (#2595), AMPKa1 (#2795), AMPKa2 (#2757), phospho-(Ser/Thr) AMPK substrate (#5759), mouse anti-phosphoATM (pS1981, #4526), phospho-Chk1 (#2360), and Chk2 (#3440) were purchased from Cell Signaling Technology. Mouse anti-tubulin (05-829) and H2AX (05-636) were purchased from Millipore. Mouse actin (ab8227), p53 (SC-126), green fluorescent protein (GFP; 632381), and rabbit anti-WIP1 (A300-664A) were purchased from Abcam, Santa Cruz, Clontech Laboratories, and Bethyl Laboratories, respectively. HA-horseradish peroxidase (HRP) (11-814-150-001) and c-Myc-HRP (12-013-819-001) were purchased from Roche. Phospho-WIP1 Ser85 antibody was raised against a peptide containing the phospho-WIP1 sequence (REARDPLPDAGA[pS]PAPSRC). Monoclonal HIPK2 antibodies were previously described (Isono et al., 2006). Animals All experiments were performed in accordance with the NIH and the Kyung Hee University guidelines for Laboratory Animal Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the Kyung Hee University. Cell Culture and Ionizing Radiation HeLa and HEK 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HCT116 p53+/+ and HCT116 p53 / cells were grown in RPMI-1640 medium supplemented with 10% FBS. Wild-type HIPK2 and KO MEFs were generated from mouse embryos at embryonic day 13.5 (E13.5) using standard procedures and cultured in DMEM media with 10% FBS. Cells were exposed to 10 Gy radiation

384 Molecular Cell 51, 374–385, August 8, 2013 ª2013 Elsevier Inc.

with a J.L. Shepherd 137Cs radiation source. Unless otherwise indicated, cells were exposed to 10 Gy g-irradiation. In Vitro Binding Assay, Liquid Chromatography-Tandem Mass Spectrometry Analysis, and Cell-Cycle Checkpoint Assay Other materials and methods are given in the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and seven figures and can be found with this article online at http://dx.doi. org/10.1016/j.molcel.2013.06.010. ACKNOWLEDGMENTS We thank William G. Dunphy, Seok-Geun Lee, Dae-Sik Lim, and Yongsok Kim for commenting on the manuscript. We also thank Kyung-Sup Kim for providing reagents. This work was supported in part by a grant from the National R&D program for Cancer Control, Ministry of Health & Welfare (2011-1120160 to C.Y.C.), the Ubiquitome Research Program (20120006126 to C.Y.C.), and the Mid-career Researcher Program through an NRF grant (2009-0085548 to C.Y.C.). We also acknowledge support from the Proteogenomic Research Program (2012M3A9B9036679 to C.L.) funded by the Korean Ministry of Education, Science and Technology. Received: October 25, 2012 Revised: April 28, 2013 Accepted: June 11, 2013 Published: July 18, 2013


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