1. Volume 24, Issue 1, Pages 77-89 (October 2006)
Phosphorylation-Dependent Control of Pc2 SUMO E3 Ligase Activity by Its Substrate Protein HIPK2 
Ana Roscic, Andreas Möller, Marco A. Calzado, Florian Renner, Verena C. Wimmer, Ekaterina Gresko, Katharina Schmid Lüdi, M. Lienhard Schmitz 
Molecular Cell 
Volume 24, Issue 1, Pages (October 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Pc2 Interacts with HIPK2 and Acts as a HIPK2 SUMO E3 Ligase
(A) Flag-tagged HIPK2 proteins were coexpressed with Myc-Pc2, Myc-PIASy, and limiting amounts of GFP-SUMO-1 in 293T cells as shown. Equal amounts of protein contained in cell lysates were analyzed by western blotting (WB) with appropriate antibodies for the occurrence and sumoylation of HIPK2 and the expression of Pc2, PIASy, and SUMO-1.
(B) Flag-HIPK2 was coexpressed with limiting amounts of GFP-SUMO-1 in the absence or presence of increasing amounts of expression vectors encoding Myc-Pc2 or Myc-PIASy as shown. Cells were lysed after 30 hr and analyzed for HIPK2 sumoylation and protein expression as above.
(C) HIPK2 and HIPK2 K25A that is mutated in the sumoylation site were 35S labeled by in vitro translation and incubated at the indicated combinations with recombinant Aos1/Uba2, Ubc9, SUMO-1, and increasing amounts of purified and recombinant (rec.) Pc2 for 90 min at 30°C. Proteins were separated by SDS-PAGE, and the migration of HIPK2 was analyzed by autoradiography.
(D) 293T cells were transfected with expression vectors for T7-tagged Pc2 in combination with various Flag-tagged HIPK2 constructs. Thirty hours after transfection, an aliquot of the cells was harvested and tested for the adequate expression of the transfected proteins, while the remaining cells were treated with a protein crosslinker and lysed under denaturing conditions. After purification of Pc2 and the interacting proteins on Ni-NTA agarose columns (Qiagen), the eluted proteins were further analyzed by immunoblotting.
Molecular Cell  , 77-89DOI: ( /j.molcel )
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3. Figure 2
Colocalization between HIPK2 and Pc2 Revealed by Confocal Microscopy and Three-Dimensional Reconstruction
(A) Colocalization of GFP-HIPK2 and T7-Pc2 in U2OS cells. Chromosomal DNA was stained with DAPI, and the merged images indicate colocalization in yellow color.
(B) Maximum projection of a confocal stack. The right part shows serial images for HIPK2 (green) and Pc2 (red) in 120 nm steps along the z axis.
(C) The three-dimensional shape of the nuclear bodies was visualized by surface rendering. The two types of speckles showing perfect (I) or imperfect (II) colocalization are shown in the boxed areas.
(D) High-resolution three-dimensional surface rendering for HIPK2 (rHIPK2, blue) and Pc2 (rPc2, yellow) in speckles I and II. The bar corresponds to 0.5 μm.
Molecular Cell  , 77-89DOI: ( /j.molcel )
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4. Figure 3
Pc2 and SUMO-1 Modification Mediate Relocalization of HIPK2 to HIPK Domains
(A) Cells expressing Flag-HIPK2 and the indicated combinations of vectors for GFP-SUMO-1 and T7-Pc2 were separated into NP40-soluble (“N”) and insoluble (“I”) fractions, which were further analyzed by immunoblotting with α-Flag antibodies for the detection of HIPK2 and α-T7 antibodies for Pc2. The lower part shows the input, as revealed by direct lysis of cells in SDS sample buffer.
(B) The experiment was done as in (A) with the exception that HIPK2 K25A was expressed instead of HIPK2.
(C) The experiment was performed as in (A) using Ubc9 instead of Pc2.
(D) The fractionation experiment was done using HIPK2 K25A and the indicated plasmids as shown.
(E) U2OS cells transfected to express GFP-HIPK2 K25A and T7-Pc2 were analyzed by confocal microscopy for the distribution of both proteins. Areas of overlapping localization are shown in yellow, and chromosomal DNA was stained with DAPI.
Molecular Cell  , 77-89DOI: ( /j.molcel )
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5. Figure 4 HIPK2 Phosphorylates Pc2
(A) T7-Pc2 was expressed either alone or together with HIPK2, followed by cell lysis and separation of phosphorylated and unphosphorylated proteins using the Qiagen PhosphoProtein Purification Kit. Fractions containing unphosphorylated and phosphorylated proteins were analyzed by western blotting for the occurrence of HIPK2 and Pc2. The phosphorylated and hyperphosphorylated forms of Pc2 are indicated by arrows, and two different exposure times are shown. The lower part shows the input material from cells directly lysed in SDS sample buffer.
(B) The experiment was done as in (A) with the exception that HIPK2 K221A was used.
(C) Cells were transfected to express Pc2 alone or in combination with HIPK2. One-half of the cells were lysed in the presence of phosphatase inhibitors. The other half was lysed in the same high-salt buffer lacking NaF and Na vanadate but containing λ-phosphatase. The electrophoretic mobility of Pc2 was determined by reducing SDS-PAGE followed by immunoblotting.
(D) 293T cells were transiently transfected with Flag-HIPK2 or kinase-inactive Flag-HIPK2 K221A. The kinases were immunoprecipitated using α-Flag antibodies and tested by western blotting for correct expression of HIPK2 (data not shown) and by immune complex kinase assays for phosphorylation of recombinant and purified GST-Pc2 or GST proteins. In vitro phosphorylation of GST-Pc2 was analyzed by SDS-PAGE followed by autoradiography. Coomassie staining of the gel confirms adequate expression of recombinant proteins.
(E) GFP-HIPK2 K221A and T7-Pc2 were coexpressed and analyzed by immunofluorescence. Note that both proteins are excluded from the nucleoli.
Molecular Cell  , 77-89DOI: ( /j.molcel )
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6. Figure 5 The HIPK2 Kinase Function Affects Pc2 Sumoylation
(A) Cells were transfected with expression vectors encoding Flag-HIPK2 or Flag-HIPK2 K221A together with GFP-SUMO-1 or T7-Pc2 as shown. Protein extracts were analyzed for the SUMO-1 modification of HIPK2 or Pc2 by immunoblotting. The slower electrophoretic mobility of HIPK2 is attributable to strong autophosphorylation.
(B) Cells were transfected to express HIPK2, HIPK2 K221A, or HIPK2 K25A along with GFP-SUMO-1 and T7-Pc2 as shown. One aliquot of the cells was directly lysed and checked for appropriate protein expression (lower part), while the remaining cells were lysed under denaturing conditions, followed by purification of Pc2 by chromatography on Ni-NTA agarose. Eluted proteins were analyzed for Pc2 sumoylation by western blotting using anti-SUMO-1 antibodies (upper).
(C and D) (C) U2OS cells transfected to express GFP-SUMO-1 and T7-Pc2 or (D) GFP-SUMO-1 and Flag-HIPK2 were stained with DAPI and analyzed for protein localization by immunofluorescence microscopy.
Molecular Cell  , 77-89DOI: ( /j.molcel )
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7. Figure 6 DNA Damage Induces Sumoylation of HIPK2 and Pc2
(A) 293T cells transfected to express Pc2 and GFP-SUMO-1 along with various combinations of HIPK2 or HIPK2 K221A were treated with adriamycin (0.5 μg/ml) as shown. Cell lysates were analyzed by immunoblotting for protein phosphorylation and sumoylation. The lower part shows a quantitative analysis of HIPK2 sumoylation.
(B) The experiment was done as in (A) with the exception that HIPK2 and HIPK2 K25A were compared.
(C) Cells transfected to express His-SUMO-1 were treated for 12 hr with adriamycin (0.5 μg/ml) as shown. One aliquot of cells was lysed in NP40 buffer to ensure appropriate expression of HIPK2 and equal loading. Note that sumoylation of proteins cannot be seen in NP40 extracts. The remaining cells were lysed under denaturing conditions, followed by chromatography on Ni-NTA agarose. Eluted proteins were further analyzed by western blotting for sumoylation of endogenous HIPK2 and expression of SUMO-1 as shown.
(D) Cells were transfected with expression vectors for Flag-HIPK2, GFP-SUMO-1, and T7-Pc2. Twenty-four hours after transfection, cells were treated with the kinase inhibitor SB (40 μM) for another 24 hr. Cell lysates were analyzed by immunoblotting with appropriate antibodies for the occurrence and sumoylation of HIPK2 and Pc2. The short exposure time (upper) of the Pc2 western blot is more suitable for detection of differences in phosphorylation, while the long exposure (lower) time allows observation of sumoylation.
(E) Cells expressing HIPK2 and GFP-SUMO-1 together with increasing amounts of T7-Pc2 or the T7-Pc2 T495A point mutant were lysed. Western blotting was used to reveal sumoylation and phosphorylation of HIPK2 and Pc2.
Molecular Cell  , 77-89DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7
Effect of Sumoylation on the Transcriptional Activity of HIPK2
(A) Cells were transfected with a luciferase reporter gene controlled by the human bax promoter and the indicated combinations of HIPK2 or HIPK2 K25A either alone or together with GFP-SUMO-1 and T7-Pc2 as displayed. Activation of bax-luc by HIPK2 and HIPK2 K25A alone was set as 100%, respectively. These transcriptional activities were diminished upon expression of GFP-SUMO-1 and/or Pc2, and fold reduction is shown. Error bars show standard deviations from three experiments performed in triplicate.
(B) A luciferase reporter gene controlled by three Gal4 DNA binding sites was cotransfected with vectors encoding the DNA-binding domain of the Gal4 or increasing amounts of Gal4-HIPK2 or Gal4-SUMO-1-HIPK2 fusion proteins. Transcriptional activity of Gal4 alone was arbitrarily set as 1, and fold induction by Gal4-HIPK2 and fold repression by Gal4-SUMO-1-HIPK2 are shown. Error bars show standard deviations from three experiments performed in duplicate.
(C) 293T cells transfected to express Flag-HIPK2 and T7-Pc2 were subjected to ChIP analysis using the indicated specific and control antibodies. Immunoprecipitates from each sample were analyzed by PCR with specific primers. Input represents the PCR product from chromatin obtained before immunoprecipitation. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining as shown.
(D) Schematic drawing summarizing the molecular mechanisms that relay DNA damage-induced HIPK2 activation to the phosphorylation of Pc2 and enhanced HIPK2 sumoylation.
Molecular Cell  , 77-89DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions