1. Volume 46, Issue 4, Pages 472-483 (May 2012)
A Redox-Regulated SUMO/Acetylation Switch of HIPK2 Controls the Survival Threshold to Oxidative Stress 
Laureano de la Vega, Inna Grishina, Rita Moreno, Marcus Krüger, Thomas Braun, M. Lienhard Schmitz 
Molecular Cell 
Volume 46, Issue 4, Pages (May 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 46, 472-483DOI: (10.1016/j.molcel.2012.03.003)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 HIPK2 Is Modified by Acetylation
(A) 293T cells were transiently transfected to express the indicated Flag-tagged kinases along with YFP-tagged CBP as shown. A fraction of the cell lysates was tested for the correct expression of the transfected CBP by western blotting, as shown in the lower part. Equal amounts of protein in the remaining extracts were used for IP of the kinases with anti-Flag antibody. After elution of bound proteins in 1× SDS sample buffer, protein acetylation was revealed by western blotting (WB) using an antibody recognizing acetylated lysines. The star indicates a nonspecific band.
(B) 293T cells were transfected with Flag-tagged HIPK2 and the indicated acetyltransferases. One part of the lysates was used as an input and loading control (lower), whereas the remaining extract was used for IP of HIPK2 and western blot analysis to detect its acetylation.
(C) 293T cells were transfected with expression vectors for HIPK2, increasing amounts of CBP, and a mutant thereof with a defect HAT activity (CBPΔHAT). Expression of the transfected proteins and HIPK2 acetylation was analyzed as in (B).
(D) 293T cells were treated for 12 hr with TSA and lysed. Following IP of acetylated proteins with an acetyl-lysine-specific antibody and western blotting, endogenous HIPK2 was detected with a specific antibody.
(E) Schematic representation of the various HIPK2 domains and the acetylated lysines. See also Figure S1 and Table S1.
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4. Figure 2 HIPK2 Acetylation Prevents Localization in Nuclear Speckles
(A) 293T cells were transfected to express CBP along with HIPK2 wild-type or mutants where the indicated lysines were mutated to arginines. Western blots were performed with antibodies recognizing the indicated acetylation sites of HIPK2 as shown.
(B) U2OS cells were lentivirally transduced to express a HIPK2-specific shRNA or a scrambled control. Three days later, cells were treated for 6 hr with TSA, and immunofluorescence with Ac-NLS1, Ac-966-, or HIPK2-specific antibodies was performed; nuclear DNA was stained with Hoechst. The percentages of cells showing the displayed localizations in this experiment and all other immunofluorescence experiments are listed in Table S2.
(C) U2OS cells transfected to express HA-tagged CBP or p300 were analyzed by indirect immunofluorescence for localization of the expressed HATs and endogenous acetylated HIPK2. Chromosomal DNA was stained with Hoechst, and the merged images indicate colocalization in yellow color.
(D) U2OS cells expressing GFP-HIPK2 were treated for 6 hr with TSA as shown.
(E) The experiment was done as in (D) with the exception that acetylated HIPK2 was stained with anti-Ac-HIPK2 antibodies. See also Figure S2.
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5. Figure 3 HDAC3 Deacetylates HIPK2
(A) U2OS cells were lentivirally transduced to express the indicated shRNAs. Three days later, they were incubated for 6 hr with TSA or nicotinamide. Acetylation of endogenous HIPK2 was revealed by immunoblotting with the use of anti-Ac-HIPK2 antibodies.
(B) The experiment was done as in (A), but cells were pretreated with Apicidin (IC50 for HDAC3 = 43 nM and for HDAC2 = 120 nM), MS-275 (IC50 for HDAC1 = 0.3 μM and for HDAC3 = 8 μM), and Tubastatin (specific for HDAC6) as shown.
(C) 293T cells were transfected with expression vectors for HIPK2, CBP, HDAC2, and HDAC3 as indicated. HIPK2 was immunoprecipitated from cell lysates, and its acetylation was measured with an antibody recognizing acetylated lysines.
(D) 293T cells were transfected with vectors for a HDAC3-specific shRNA or the empty vector as a control, followed by selection of transfected cells with puromycin for 3 days. Cell lysates were tested for efficient HDAC3 knockdown and acetylation of endogenous HIPK2.
(E) Lysates from 239T cells were used for IP with rabbit anti-HDAC3 antibodies or controls, followed by detection of HDAC3 and coprecipitated HIPK2 by immunoblotting. The lower part shows the input material.
(F) U2OS cells were stained for endogenous HIPK2 and HDAC3 with specific antibodies and analyzed by indirect immunofluorescence to reveal the localization HIPK2 and HDAC3. See also Figure S3.
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6. Figure 4 SUMOylation of HIPK2 Antagonizes Its Acetylation
(A) Flag-tagged HIPK2 and a non-SUMOylatable mutant (HIPK2 K25A) were expressed in 293T cells along with CBP and increasing amounts of GFP-SUMO1. One part of the lysates was used as an input and loading control (lower), whereas the remaining extract was used for IP of HIPK2 and western blot analysis to detect its acetylation. The arrows indicate HIPK2 SUMOylation, as revealed by the slower migrating band.
(B) 293T cells expressing His-tagged HIPK2, HA-CBP, and GFP-SUMO1 were harvested and either directly lysed in 1× SDS sample buffer for the input controls (lower) or in guanidine-HCl containing buffer allowing the enrichment of the SUMOylated kinase under denaturing conditions on Ni-NTA columns. The eluates were run on a SDS gel, blotted to a membrane, and then analyzed for SUMOylation and acetylation of HIPK2 by immunoblotting with specific antibodies. The positions of molecular weight markers are indicated.
(C) 293T cells were transfected to express HIPK2 or a SUMO1-HIPK2 fusion protein either alone or in combination with CBP as shown. Lysates were tested by immunoblotting for protein expression and HIPK2 acetylation as shown.
(D) 293T cells were transfected with vectors directing the expression of a HDAC3-specific shRNA or a scrambled control. After 3 days, cells were retransfected to express HIPK2, CBP, GFP-SUMO, and the HDAC3 shRNA. Cells were directly lysed in 1× SDS sample buffer and characterized for HDAC3 knockdown and HIPK2 modification. Note that in the absence of HDAC3, the SUMOylated form of HIPK2 is also acetylated.
(E) Cells were transfected with expression vectors encoding HIPK2, SUMO1-HIPK2, or HDAC3 as shown. Lysates were prepared, followed by analysis of protein expression (lower) and by analysis of protein/protein interactions using coIP (upper).
(F) Cells expressing Flag-tagged HDAC3 were lysed, and the lysate was further analyzed by a GST pull-down experiment. Of the lysate, 10% was used as an input control. See also Figure S4.
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7. Figure 5 H2O2 Triggers De-SUMOylation and Acetylation of HIPK2
(A) U2OS cells virally transduced to express a HIPK2-specific shRNA or a control were treated with 400 μM H2O2 for 6 hr. Cells were directly lysed in 1× SDS buffer, and extracts were analyzed for acetylation of endogenous HIPK2 by western blotting.
(B) 293T cells were treated with 100 nM of TSA, followed by the addition of increasing concentrations of H202 (100, 300, and 600 μM) for 6 hr. Cell lysates were analyzed by immunoblotting to determine HIPK2 acetylation as shown.
(C) 293T cells were transfected with an expression vector encoding Flag-HIPK2 and GFP-SUMO. The next day, cells were treated for 6 hr with increasing H2O2 concentrations (100, 200, and 300 μM). Cells were directly lysed in 1× SDS sample buffer to maintain HIPK2 SUMOylation and analyzed by western blotting.
(D) 293T cells expressing epitope-tagged versions of HIPK2 and HDAC3 were treated for 6 hr with 300 and 600 μM of H2O2, followed by treatment of cells with a membrane-permeable crosslinker to allow covalent coupling of proteins being in very close proximity. One part of the cell extract was used for coIP using an anti-HA antibody. The coprecipitating HDAC3 protein was detected with anti-GFP antibodies; the lower part shows the input material.
(E) HIPK2 was expressed along with HA-CBP or GFP-SUMO1 to trigger or antagonize acetylation, respectively. 293T cells were treated with increasing concentrations (up to 400 μM) of H2O2. A fraction of the cells was lysed in IP buffer to determine HIPK2 acetylation by IP followed by immunoblotting with an acetyl-lysine-specific antibody. The lower part shows the input material, as revealed by direct lysis of cells in SDS sample buffer to preserve SUMOylation of HIPK2.
(F) 239T cells transfected to express HIPK2 or SUMO1-HIPK2 were treated for 6 hr with 400 μM H2O2. HIPK2 expression and acetylation were revealed by immunoblotting as shown. See also Figure S5.
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8. Figure 6 HIPK2 Acetylation Restricts Its Apoptosis-Inducing Properties
(A) HIPK2 wild-type or HIPK2 10KR was expressed along with increasing amounts of CBP in 293T cells. One part of cell lysates was used to immunoprecipitate HIPK2 with an anti-Flag antibody and to test its acetylation by an acetyl-lysine antibody as shown. The lower part shows the input material.
(B) The indicated HIPK2 mutants were coexpressed in 293T cells with the HIPK2 substrate protein Siah2 RM (ring mutant), which was mutated in the RING domain to prevent HIPK2 ubiquitination and degradation. Cell lysates were analyzed by immunoblotting for Siah2 phosphorylation as detected by a phospho-specific antibody and by the occurrence of the upshifted, phosphorylated Siah2 fraction. The band corresponds to phosphorylated SIAH2. The antibody recognizes SIAH2 phosphorylated at serine 28.
(C) Equal numbers of HIPK2 knockout cells stably transduced with the indicated HIPK2 mutants or empty vector control were grown on dishes. Cells were treated for 24 hr with the indicated concentrations of H2O2 followed by a medium exchange. The surviving cells were further grown to colonies and stained with crystal violet.
(D) The indicated cell lines were treated for 24 hr with 400 μM of H2O2, and cell death was quantified by propidium iodide staining in a flow cytometer. Error bars show SEM from two independent experiments performed in triplicate.
(E) The cells lacking HIPK2 or reconstituted with the indicated HIPK2 variants were analyzed by quantitative analysis of gene expression by real-time PCR as shown. In order to facilitate comparison, full gene activation was arbitrarily set as 100%. Experiments were performed in triplicates; error bars display SEMs.
(F) The indicated cells were incubated for 15 min in the presence of 10 μM DCFH-DA. Fluorescence of DCF was detected and analyzed on a FACSCalibur flow cytometer using CellQuest analysis software (Becton Dickinson). Data are depicted by mean fluorescence intensity (MFI); error bars show SEMs from two independent experiments performed in triplicate. See also Figure S6.
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9. Figure 7 Schematic Summary of the Results
For more details, please refer to the Discussion. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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