1. Volume 51, Issue 3, Pages 374-385 (August 2013)
WIP1, a Homeostatic Regulator of the DNA Damage Response, Is Targeted by HIPK2 for Phosphorylation and Degradation 
Dong Wook Choi, Wooju Na, Mohammad Humayun Kabir, Eunbi Yi, Seonjeong Kwon, Jeonghun Yeom, Jang-Won Ahn, Hee-Hyun Choi, Youngha Lee, Kyoung Wan Seo, Min Kyoo Shin, Se-Ho Park, Hae Yong Yoo, Kyo-ichi Isono, Haruhiko Koseki, Seong-Tae Kim, Cheolju Lee, Yunhee Kim Kwon, Cheol Yong Choi 
Molecular Cell 
Volume 51, Issue 3, Pages (August 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 51, 374-385DOI: (10.1016/j.molcel.2013.06.010)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 HIPK2 Is a Protein Kinase that Phosphorylates WIP1
(A) HCT116 (p53+/+) and HCT116 (p53−/−) cells were exposed to γ-irradiation in the presence or absence of MG132, and cells were analyzed for WIP1 and p53 levels at the indicated time points after γ-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 γ-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 γ-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.
Molecular Cell  , DOI: ( /j.molcel )
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4. 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 γ-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 γ-irradiation, followed by immunoblotting with anti-WIP1 and anti-HIPK2 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 anti-ubiquitin (Ub) antibody.
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5. 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 γ-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 γ-32P-labeled 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.
Molecular Cell  , DOI: ( /j.molcel )
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6. 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 γ-irradiation, and lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies.
(C) HCT116 cells were either untreated or γ-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 γ-irradiation. Cells were harvested at the indicated time points, and lysates were immunoprecipitated with anti-HIPK2 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 γ-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 γ-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, siAMPKα1, or two independent siAMPKα2 (#1 or #2), and cells were analyzed with the same procedure as in (F).
(H) H1299 cells were transfected with control siRNA or siAMPKα2 followed by γ-irradiation, and they were harvested at the indicated time points after CHX treatment. Cell lysates were analyzed by immunoblotting with anti-WIP1 and anti-AMPKα2 antibodies.
(I) H1299 cells were transfected with control siRNA, siAMPKα1, or siAMPKα2 in combination with expression plasmids encoding HA-HIPK2, and cells were left untreated or γ-irradiated. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with the indicated antibodies.
(J) HCT116 cells were transfected with wild-type HA-HIPK2, and cells were left untreated or exposed to γ-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 γ-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 γ-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.
Molecular Cell  , DOI: ( /j.molcel )
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7. 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 γ-irradiation, and treated with wortmannin 3 hr prior to fixation of cells for immunostaining with anti-γ-H2AX antibody. HIPK2 depletion by siRNA was confirmed by immunoblotting with anti-HIPK2 antibody. The number of γ-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 γ-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 γ-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 γ-irradiation and harvested at the indicated time points. Cell lysates were immunoblotted with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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8. 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 γ-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 γ-irradiation (16 Gy) was measured by FACS analysis. Fold reduction of BrdU incorporation following γ-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 γ-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 γ-irradiation. The tissue extracts were analyzed by immunoblotting with anti-HIPK2, anti-WIP1, and anti-γ-H2AX antibodies.
(F) Wild-type and hipk2 heterozygous mice were whole-body irradiated (6 Gy), and the survival curves are shown.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Schematic Summary of HIPK2-Mediated WIP1 Regulation in Unstressed and γ-Irradiated Cells
(A) Molecular diagram for HIPK2-mediated WIP1 phosphorylation and proteasomal degradation and AMPKα2-mediated inhibition of WIP1 phosphorylation by HIPK2.
(B) Dynamics of relative WIP1 levels before or after γ-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 AMPKα2 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.
Molecular Cell  , DOI: ( /j.molcel )
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