Regulation of p53 activity by its interaction with homeodomain - Nature

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Regulation of p53 activity by its interaction with homeodomaininteracting protein kinase-2 Thomas G. Hofmann*†, Andreas Möller*, Hüseyin Sirma†, Hanswalter Zentgraf‡, Yoichi Taya§, Wulf Dröge*, Hans Will† and M. Lienhard Schmitz*¶|| *Division of Immunochemistry (G0200) and ‡Division of Applied Tumour Virology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany †Department of General Virology, Heinrich-Pette-Institut für experimentelle Virologie und Immunologie, Martinistrasse 52, 20251 Hamburg, Germany §Radiobiology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan ¶e-mail: [email protected] ||Current address: Department for Chemistry and Biochemistry, Freiestrasse 3 CH-3012, Bern, Switzerland

Published online: 10 December, DOI: 10.1038/ncb715

Transcriptional activity of p53, a central regulatory switch in a network controlling cell proliferation and apoptosis, is modulated by protein stability and post-translational modifications including phosphorylation and acetylation. Here we demonstrate that the human serine/threonine kinase homeodomain-interacting protein kinase-2 (HIPK2) colocalizes and interacts with p53 and CREB-binding protein (CBP) within promyelocytic leukaemia (PML) nuclear bodies. HIPK2 is activated by ultraviolet (UV) radiation and selectively phosphorylates p53 at Ser 46, thus facilitating the CBP-mediated acetylation of p53 at Lys 382, and promoting p53-dependent gene expression. Accordingly, the kinase function of HIPK2 mediates the increased expression of p53 target genes, which results in growth arrest and the enhancement of UV-induced apoptosis. Interference with HIPK2 expression by antisense oligonucleotides impairs UV-induced apoptosis. Our results imply that HIPK2 is a novel regulator of p53 effector functions involved in cell growth, proliferation and apoptosis.

he tumour-suppressor protein p53 is important in the cellular response to genotoxic damage. In non-stressed cells, p53 is kept silent and at low levels after association with Mdm2, which represses transcription when associated with p53 and promotes its proteolytic degradation1–4. Because mdm2 is a p53 target gene, both proteins are mutually regulated in a negative feedback loop5. Adverse agents such as irradiation with UV and γ-rays, withdrawal of growth factors, hypoxia and oncogenes such as Ras induce signalling events that result in the transient stabilization of the p53 protein and the onset of p53 function as a transcriptional activator or repressor within the cell nucleus6,7. The induction of p53 involves several mechanisms including post-translational modifications such as phosphorylation and acetylation8. DNAdamage-induced phosphorylation of serines and threonines within the p53 amino terminus contributes to p53 stability by preventing Mdm2 from binding and by rendering p53 more resistant to Mdm2 (refs 9–11). Five serines in the carboxy-terminal portion and seven serines and one threonine within the N-terminal 46 amino acids are known to be inducibly phosphorylated12, but the exact order of phosphorylations and their specific contribution to p53 effector function remain to be elucidated. For example, Ser 6, Ser 9, Ser 15, Ser 20 and Ser 37 are phosphorylated in response to DNA damage, probably by protein kinases including ATM, Chk2, DNA-PK and ATR13–16. Phosphorylation of Ser 15 is observed only in response to DNA damage17, but not after p53 activation in response to E1A expression18, showing that the specific roles of individual phosphorylation sites depend on the stimulus and probably the cell type. In addition, phosphorylation of Ser 46 is functionally important, because mutation of this site within the p53 protein decreases sensitivity towards UV-induced apoptosis19,20. Acetylation of the p53 protein occurs in response to many

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different p53-activating stimuli in vivo21,22. Lys 373 and Lys 382 are inducibly acetylated by cAMP-response-element-binding (CREB)binding protein (CBP) and the closely related p300 protein21,23, whereas Lys 320 is acetylated in vitro by the acetylase P/CAF (p300/CBP-associated factor) 24. Acetylation stimulates DNA-binding activity of p53 in vitro and thus enhances p53-dependent gene expression21,23, p53-mediated apoptosis and growth arrest25, whereas overexpression of a dominant-negative form of p300 counteracts p53 function. The activation of p53 not only involves post-translational modifications and protein stabilization, but is also linked to localization into distinct subnuclear structures: expression of the promyelocytic leukaemia (PML) protein isoform PML3, Rasderived signals, γ-irradiation or treatment of cells with UV radiation and As2O3 lead to the recruitment of p53 into PML nuclear bodies (PML-NBs)26–29. Several lines of evidence support the concept that PML-NBs are subnuclear locations required for p53 activity: PMLNB localization of p53 and its interaction with other PML-NB proteins such as CBP and PML stimulate p53-dependent transcription and effector functions26,27,29. PML−/− cells fail to recruit p53 into PML-NBs and to induce p53-dependent gene expression and Rasinduced senescence27–29. Transcriptional activation by p53 within the PML-NBs is augmented by the acetylation of Lys 382 and is, in part, PML-dependent29. Finally, PML mutants that are incapable of forming PML-NB structures but still associate with p53 fail to trigger p53-dependent transcription28. Although PML-NB localization and phosphorylation are important for p53 function, a kinase located in PML-NBs has not yet been identified. We have recently cloned the human serine/threonine kinase HIPK2 (ref. 30), a kinase described to act as a corepressor for homeodomain transcription factors31. Because HIPK2 from

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Figure 1 PML3 recruits HIPK2 to PML-NBs. a, Representative images of U2OS cells stained for endogenous HIPK2 (green) and PML (red) by indirect immunofluorescence. Overlapping localization is shown in yellow. b, U2OS cells were transiently transfected with an expression vector for PML3 and analysed for endogenous HIPK2 (green) and PML3 (red) distribution; overlapping localization is shown in the third panel. c, H1299 cells were transfected with an expression vector encoding Flag-tagged HIPK2 and analysed for HIPK2 (green) and endogenous PML (red) by indirect immunofluorescence; arrowheads in the third panel point to yellow dots showing overlapping localization. d, H1299 cells were transfected with of vectors encoding Flag-HIPK2 (green) and PML3 (red); overlapping localization is shown in the third panel. In b–d, the fourth panels show nuclear DNA stained with DAPI.

mouse31 and hamster32 localizes to distinct subnuclear dots, we were prompted to investigate whether human HIPK2 is a component of PML-NBs. Our results show that HIPK2 associates with p53 and CBP and is recruited together with its interactors into PMLNBs, where HIPK2 contributes to p53 activation in response to UV radiation.

Results HIPK2 is recruited to PML-NBs where it localizes together with p53 and CBP. To study the subcellular distribution of human HIPK2, U2OS cells were analysed for localization of endogenous HIPK2. Immunofluorescence staining revealed most HIPK2 to be in subnuclear speckles and a small amount of the kinase in the nucleoplasm (Fig. 1a). Simultaneous staining with the endogenous PML protein showed that most HIPK2 had a distinct localization to PML, and a minor fraction of HIPK2 was contained in PML-NBs (Fig. 1a). Because the PML splice variant PML3 is capable of recruiting proteins such as p53 specifically into PML-NBs27, we investigated the effect of PML3 expression on the subnuclear localization of endogenous HIPK2 in U2OS cells. PML3 increased the size of PML-NBs and efficiently translocated HIPK2 into this subnuclear structure (Fig. 1b), whereas expression of the PML splice variant PML-L failed to recruit HIPK2 to PML-NBs (data not shown). To test whether physiological levels of ectopically expressed HIPK2 show a similar intranuclear distribution pattern to that of the endogenous kinase, H1299 cells were transiently transfected with a vector encoding Flag-tagged HIPK2. Immunofluorescence staining revealed predominant localization of 2

HIPK2 in discrete subnuclear speckles that were colocalizing only partly with the endogenous PML protein (Fig. 1c). In addition, ectopically expressed HIPK2 was almost completely recruited into PML-NBs on expression of PML3 (Fig. 1d), showing that the endogenous and overexpressed HIPK2 proteins behave similarly. Because the p53 protein and its coactivator CBP can localize within PML-NBs33,34, we investigated whether HIPK2 can be recruited simultaneously to this subnuclear compartment favorably for p53 activity. Triple stainings revealed that H1299 cells transfected with green fluorescent protein (GFP)-labelled p53, CBP and PML3 showed a largely identical subcellular localization of all three proteins (Fig. 2a). Furthermore, cells coexpressing HIPK2, GFP–p53 and CBP displayed almost identical staining patterns for these proteins, in the presence of PML3 (Fig. 2b). PML3, HIPK2 and CBP proteins expressed together showed the same subnuclear distribution for all three proteins in NBs (Fig. 2c). This demonstrates that PML3 enhances the recruitment of HIPK2, p53 and CBP into PML-NBs. In the absence of PML3 expression, p53 and the p53 coactivator CBP showed both a speckled and diffuse microgranular nuclear staining that also partly overlapped with that of HIPK2 localized in NBs (data not shown). Localization of endogenous HIPK2 and p53 proteins together in PML-NBs was also enhanced in response to a different stimulus. Treatment of U2OS cells with UV irradiation and As2O3 increased the localization of both endogenous proteins together (Fig. 2d), arguing for the physiological relevance of these findings. Under these conditions, the endogenous HIPK2/PML and PML/p53 (Fig. 2d) and CBP/PML (Fig. 2e) proteins showed increased colocalization, demonstrating that genotoxic stress leads to the recruitment of these proteins to PML-NBs. HIPK2 binds to p53 in vitro and in vivo. To examine a possible direct interaction of p53 and HIPK2, glutathione S-transferase (GST) pull-down experiments were performed. The wild-type form of HIPK2, a kinase-deficient point mutant (HIPK2-K221A) and various deletion mutants were 35S-labelled by translation in vitro and tested for binding to a purified GST–p53 fusion protein (Fig. 3a). Both wild-type HIPK2 and HIPK2-K221A were found to associate with GST–p53 in vitro. A HIPK2 deletion mutant lacking the C terminus failed to interact with p53. In contrast, the C-terminal portion of HIPK2 was sufficient for p53 binding, identifying the HIPK2 C terminus as the p53-interacting region. The p53 domain mediating interaction with HIPK2 was mapped by measuring HIPK2 binding to various portions of p53 fused to GST. These experiments revealed that HIPK2 interacts directly with p53 amino acids 294–393 (Fig. 3b). To test whether HIPK2 and endogenous p53 also interact in vivo, Flag-tagged versions of HIPK2 and mutants thereof were expressed in HEK 293 cells. Co-immunoprecipitation experiments showed an interaction of the wild-type, kinase-inactive (data not shown) and N-terminally truncated forms of HIPK2 with endogenous p53, whereas no interaction occurred with HIPK2∆C (Fig. 3c). The association between both endogenous proteins was investigated by co-immunoprecipitation experiments. HIPK2 was immunoprecipitated from cell extracts prepared from untreated or UV/As2O3-stimulated U2OS cells. Subsequent immunoblotting revealed the occurrence of small amounts of p53 in immunoprecipitates from unstimulated U2OS cells but not from p53-deficient H1299 control cells. Extracts from UV/As2O3-treated U2OS cells contained much higher amounts of p53 in the HIPK2 immunoprecipitate, as revealed by immunoblotting with FL393 (Fig. 3d) or DO-1 (data not shown) anti-p53 antibodies. This inducible co-immunoprecipitation of the endogenous proteins was also seen in HEp2 cells (data not shown). These results show the induced physical association of the endogenous proteins under physiological conditions. HIPK2 activates p53-regulated transcription dependent on its kinase activity. To define the functional relevance of this protein–protein interaction on p53-dependent transcription, increasing amounts of HIPK2 were expressed in HEK 293 cells along with NATURE CELL BIOLOGY VOL 4 JANUARY 2002 http://cellbio.nature.com


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Figure 2 Colocalization of HIPK2 with p53, CBP and PML in PML-NBs. a, H1299 cells were transfected to express PML3 along with GFP–p53 and CBP. Triple stainings are shown; GFP–p53 (green) was detected by the intrinsic fluorescence of GFP. PML3 (blue) and CBP (red) were detected by indirect immunofluorescence. b, H1299 cells were transfected with an expression vector encoding PML3, Flag-tagged HIPK2, GFP–p53 and CBP. The indicated proteins were detected as described above. c, Flag-tagged HIPK2, CBP and PML3 were expressed in H1299

cells and analysed by indirect immunofluorescence. d, U2OS cells were stimulated with UV (30 J m−2) and 1 µM As2O3 (6 h). Endogenous HIPK2 (green), PML (blue) and p53 (red) are shown; areas of overlapping localization are indicated by arrowheads in the lower panels. e, Colocalization (yellow) of endogenous CBP (green) and PML (red) proteins in UV/As2O3-treated U2OS cells as analysed by indirect immunofluorescence. Nuclear DNA was stained with DAPI (fourth panel).

a luciferase reporter gene controlled by multimers of either intact (pG13-luc) or mutated (pMG15-luc) p53-binding sites. Dependent on the integrity of the p53 responsive elements, expression of HIPK2 resulted in a dose-dependent induction of transactivation (Fig. 4a). To measure the p53 dependence of HIPK2-mediated effects in the context of a natural p53-dependent promoter, cells expressing the temperature-sensitive p53-C135V mutant were transfected with a p21Waf1-promoter-dependent luciferase reporter gene together with vectors encoding HIPK2 or HIPK2-K221A. Transcription from the p21Waf1 promoter was induced by HIPK2 in a kinase-dependent manner exclusively at the permissive temperature (Fig. 4b). To measure the p53 dependence of this HIPK2-mediated effect by an independent experimental approach, p53-deficient SaOS-2 cells were transfected with a luciferase reporter gene controlled by the p21Waf1 promoter together with various combinations of vectors encoding p53, HIPK2 or HIPK2-K221A (Fig. 4c). Whereas HIPK2 alone failed to induce p21Waf1-dependent transcription, it triggered gene expression in the presence of p53. In contrast, the kinase-inactive version of HIPK2 had no effect on gene expression. Taken together, these results support the idea that the transcriptional effects of HIPK2 on p21Waf1 are mediated by p53. In addition, the human bax and PIG3 promoter was found to be HIPK2-inducible in a p53-dependent manner (data not shown). HIPK2 phosphorylates p53 at Ser 46 and is activated by irradiation with UV. Because the kinase function of HIPK2 is required for its effect on p53 activity, we were prompted to investigate a direct phosphorylation of p53 by HIPK2. Various portions of the p53 protein were expressed as bacterial GST fusion proteins and tested for phosphorylation by HIPK2 in immune complex kinase assays. Only the N-terminal 80 amino acids were phosphorylated by HIPK2 (see Supplementary Information, Fig. S1a–c), whereas more C-terminal portions of p53 fused to GST remained unphosphorylated (data not shown). Because kinase-inactive HIPK2 did not increase the incorporation of 32P into GST–p53(1–80), we consider phosphorylation by co-precipitating kinases to be unlikely. The phosphorylation site within p53 was determined after transfection of p53-negative H1299 cells with expression vectors for p53 in combination with HIPK2 or HIPK2-K221A, followed by an analysis of phosphorylation with the use of phospho-specific antibodies. Dependent on its kinase function, expression of

HIPK2 selectively induced Ser 46 phosphorylation in intact cells. In contrast, HIPK2 did not phosphorylate Ser 20 (Fig. 5a) or Ser 6, Ser 9 and Ser 15 (data not shown). The functionality of antibodies specifically recognizing phosphorylated Ser 15 and Ser 20 was ensured in control experiments with extracts from UV-irradiated cells (data not shown). To prove the direct phosphorylation of p53 by HIPK2, Flag-tagged versions of wild-type HIPK2, HIPK2-K221A and HIPK2∆C were produced by translation in vitro. After purification of the proteins by immunoprecipitation with anti-Flag antibodies, kinase activity was measured by in vitro kinase assays with the GST–p53 fusion protein as a substrate. HIPK2 and HIPK2∆C showed strong autophosphorylation, but only HIPK2 was able to phosphorylate its substrate protein GST–p53 (Fig. 5b). Because HIPK2∆C fails to bind p53 (see Fig. 3a), these results show that interaction of HIPK2 with p53 is necessary for phosphorylation and exclude the phosphorylation of p53 by a kinase contained in the rabbit reticulocyte lysate. The functional impact of HIPK2-mediated Ser 46 phosphorylation on the transcriptional activity of p53 was tested after the transfection of H1299 cells with a luciferase reporter fused to the p21Waf1 promoter along with expression plasmids encoding HIPK2, p53 and a p53 point mutant containing a serine to alanine change at position 46 (p53-S46A). Whereas p53 and p53-S46A alone activated the p21Waf1 promoter to the same extent, the HIPK2-stimulated activity of p53 was higher than that of p53S46A (Fig. 5c). These results show that HIPK2-mediated Ser 46 phosphorylation efficiently contributes to the transactivation potential of p53. We then tested various well-characterized p53 activating signals for their ability to induce the phosphorylation and subsequent activation of endogenous HIPK2. MCF-7 cells were either left untreated or stimulated by irradiation with UV or γ-rays, followed by the immunoprecipitation of endogenous HIPK2 and in vitro kinase assays with the known HIPK2 phosphorylation substrate myelin basic protein (MBP)30. Irradiation with UV strongly activated the phosphorylation of MBP and HIPK2, whereas γ-irradiation remained without impact on HIPK2 kinase activity (Fig. 5d). This induction was not due to elevated levels of HIPK2 in response to UV treatment (see Supplementary Information, Fig. S2). Because the phosphorylation of p53 at Ser 46 (ref. 20) and HIPK2 activity can be induced by UV,



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Figure 3 HIPK2 interacts with p53 in vitro and in vivo. a, HIPK2 variants translated In vitro and labelled with 35S were tested for binding to bacterially produced GST and GST–p53 proteins as shown. The upper panel shows schematic representations of HIPK2 and its mutants. The asterisk indicates the location of the mutated lysine in the kinase domain; numbers show the amino-acid positions. The input lane represents 50% of the material used for binding to the GST fusion protein. b, Labelled HIPK2 was assayed for binding to the indicated GST–p53 variants. The transactivation (TA), DNA-binding (DNA-BD) oligomerization (OD) and regulatory (RD) domains within the p53 protein are shown; autoradiograms from representative experiments are shown. The input lane represents 50% of the material used. c, HEK 293 cells were transfected to express various Flag-tagged HIPK2 proteins

(8 µg) as shown. After immunoprecipitation (IP) of HIPK2 with anti-Flag antibodies, co-precipitating endogenous p53 protein was detected by western blotting (WB) (upper panels). The heavy chain of the precipitating antibody (IgGH) is indicated. The blots were also tested for expression of HIPK2 variants by using anti-Flag antibodies (lower panels). The input lane represents 25% of the material used in the co-precipitation. d, U2OS and H1299 cells were left untreated or stimulated with UV (30 J m−2) and 1 µM As2O3 (6 h) as shown, and lysed. Endogenous HIPK2 was immunoprecipitated from an aliquot of the cell lysates with rabbit polyclonal anti-HIPK2 antibodies. Proteins were eluted from the washed immunoprecipitates with 1 × SDS sample buffer and analysed by western blotting for the occurrence of either p53 (upper panels) or HIPK2 (lower panels). One-quarter of the total cell lysate was added as a control.

we compared the kinetics of UV induction. Phosphorylation of p53 at Ser 46 was correlated with the induction of HIPK2 6 h after irradiation with UV (Fig. 5e), supporting the importance of HIPK2 for this phosphorylation site. HIPK2 is associated with CBP and triggers the CBP-mediated acetylation of p53. The localization of HIPK2 together with the p53 interactor CBP prompted us to examine whether HIPK2 also interacts with CBP. Immunoprecipitates of endogenous HIPK2 from lysates of U2OS or MCF-7 cells contained the CBP protein, whereas no CBP was detected in immunoprecipitates of a control antibody (Fig. 6a), showing the mutual interaction of endogenous CBP and HIPK2 proteins. The HIPK2 domain mediating this interaction was mapped in further co-immunoprecipitation experiments. The full-length form of Flag-tagged HIPK2 and a HIPK2 variant lacking the C-terminal half were expressed together with CBP in HEK 293 cells. Both proteins were readily detected in CBP immunoprecipitates from these cells (Fig. 6b). In contrast, a HIPK2 variant lacking the N terminus did not bind, mapping the interaction domain to the N-terminal portion of HIPK2. To test a possible

modulatory function of HIPK2 on the acetyltransferase activity of CBP, the acetylation of p53 expressed in p53-negative H1299 cells together with different combinations of CBP, HIPK2 and HIPK2K221A was measured by using acetylation-specific antibodies. CBP-mediated acetylation of p53 at Lys 373 and Lys 382 (ref. 24) was increased in a kinase-dependent manner after the expression of HIPK2 (Fig. 6c). A control experiment shows that a p53 protein mutated at Lys 382 was not acetylated in response to CBP/HIPK2. It has been shown24 that DNA damage-induced phosphorylation of the N terminus of p53 results in an increased affinity for the acetyltransferases CBP/p300, thus promoting the subsequent acetylation of Lys 382 within the C terminus of p53. To test whether such a mechanism also occurs in response to HIPK2-mediated p53 phosphorylation, H1299 cells were transfected to express HIPK2 and/or CBP together with p53 or p53-S46A. HIPK2/CBP-mediated acetylation occurred only in the p53 wild-type protein, whereas p53S46A remained completely unacetylated (Fig. 6d), revealing that HIPK2-mediated p53 phosphorylation at Ser 46 is required for acetylation. The functional impact of HIPK2/CBP-mediated

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Figure 4 HIPK2 activates p53-dependent promoters. a, HEK 293 cells were cotransfected with luciferase reporter constructs controlled by multimers of either intact (pG13-Luc) or mutated (pMG15-Luc) p53-binding sites and increasing amounts of HIPK2 expression vector as shown. Transactivation by the empty expression vector was arbitrarily set as 1. b, Rat embryo cl-6 cells expressing a temperature-sensitive p53 mutant (p53 C135V) were transiently transfected with p21Waf1 promoter fused to the luciferase reporter gene, in combination with 2 µg of expression vector for HIPK2 or HIPK2-K221A. Cells were further cultured for 1 d at the permissive (32 °C) or non-permissive (37.5 °C) temperature and analysed for luciferase activity. Reporter gene activation induced by empty control vector was arbitrarily set as 1. c, SaOS-2 cells were transfected to express the indicated combinations of p53 (10 ng) and 3 µg HIPK2 or HIPK2-K221A together with the p21Waf1 reporter gene. Reporter gene activation induced by p53 was arbitrarily set as 100. Bars in all experiments indicate s.d. from at least three independent experiments.

acetylation on p53-dependent gene expression was assessed in H1299 cells by reporter gene assays. Stimulation of p53-dependent transcription occurring after the simultaneous expression of p53 and CBP was further boosted by HIPK2. In contrast, the stimulatory effect of HIPK2/CBP on a p53 variant mutated at Lys 382 (p53-K382A) was impaired (Fig. 6e), showing that HIPK2/CBPmediated acetylation of this lysine augments the transcriptional activity of p53. To investigate the role of HIPK2 for UV-induced p53 acetylation, H1299 cells were transfected with an expression

vector encoding p53 together with the wild-type or kinase-inactive form of HIPK2. Untreated or UV-stimulated cells were lysed and p53 acetylation was determined by immunoblotting. UV-induced acetylation of p53 was significantly augmented by HIPK2, whereas p53 acetylation remained unaffected by HIPK2-K221A (Fig. 6f). Kinase activity of HIPK2 induces cell-cycle arrest and increases UV-induced apoptosis. We next examined whether HIPK2-mediated post-translational modifications of p53 have any consequences for p53-dependent biological functions such as the regulation of cell proliferation. Colony formation assays performed in U2OS cells revealed that the expression of HIPK2 prevented cell growth dependent on its kinase activity (Fig. 7a). The relative importance of p53 for the anti-proliferative function of HIPK2 was tested in p53deficient H1299 cells by using the same experimental approach. In comparison with U2OS cells, these p53-deficient cells showed a strongly impaired growth suppression by HIPK2 (Fig. 7b), revealing the dependence on p53 of a substantial part of the growth-suppressing function of HIPK2. However, HIPK2 still prevented colony formation of H1299 cells to a certain extent, raising the possibility of further p53-independent mechanisms triggered by this kinase. Because p21Waf1 acts as a Cdk inhibitor and thereby contributes to the control of the G1/S checkpoint35, we next determined the impact of HIPK2 on progression into S phase. HIPK2 or HIPK2K221A was expressed in U2OS cells, and bromodeoxyuridine (BrdU) incorporation was determined by immunofluorescence microscopy. HIPK2-expressing cells showed a strongly decreased percentage of cells in the S phase, as indicated by an impaired incorporation of BrdU (Fig. 7c). In contrast, kinase-deficient HIPK2 did not affect DNA synthesis, showing that HIPK2 can interfere with the cell cycle in a kinase-dependent manner. A similar experimental approach in p53-negative H1299 cells revealed increased BrdU incorporation in cells expressing HIPK2 than in p53-containing U2OS cells, and no effects on DNA synthesis in cells expressing HIPK2-K221A (Fig. 7d). To determine whether the lack of proliferation and BrdU incorporation involves an upregulation of p21Waf1 at the protein level, U2OS cells were transfected with vectors encoding p53 and/or HIPK2 and analysed for p21Waf1 by western blotting. Whereas the expression of HIPK2 alone increased p21Waf1 protein levels, the simultaneous expression of HIPK2 and p53 led to a further increase in the amount of this cell-cycle regulator (Fig. 7e). The role of HIPK2-mediated Ser 46 phosphorylation in apoptosis was investigated after the transfection of U2OS cells with enhanced GFP (EGFP)–HIPK2 and EGFP–HIPK2-K221A expression vectors. The cells were irradiated with a sublethal dose of UV (10 J m−2), which induces the phosphorylation of Ser 15 and Ser 20 but not that of Ser 46 (data not shown)20. UV-induced apoptosis was efficiently enhanced after the expression of HIPK2 in a kinasedependent manner (Fig. 8a), supporting the importance of HIPK2-mediated Ser 46 phosphorylation (in combination with the UV-triggered phosphorylation of Ser 15 and Ser 20) for UVinduced apoptosis. To investigate the relative contribution of endogenous HIPK2 for UV-mediated apoptosis, antisense morpholino oligonucleotides were used to decrease HIPK2 expression in U2OS cells (Fig. 8b). Impairment of HIPK2 expression led to a pronounced inhibition of UV-induced apoptosis (Fig. 8c) and phosphorylation of p53 at Ser 46 (Fig. 8d), thus revealing an important contribution of HIPK2 to this apoptotic pathway.

Discussion Here we identify the serine/threonine kinase HIPK2 as a novel activator of the p53 tumour suppressor protein. Colocalization studies, biochemical and functional experiments reveal a role for HIPK2 in the post-translational modification and regulation of p53 effector function. The importance of PML-NBs in the acetylation of p53 at Lys 382, in transcriptional activation and in the regulation of p53dependent senescence is evident from a variety of experimental



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Figure 5 HIPK2 is activated by UV irradiation and phosphorylates Ser 46 of p53. a, p53 (1 µg of expression vector) was expressed together with Flag-tagged HIPK2, HIPK2-K221A or empty control vector (3 µg of each) in H1299 cells. Cell lysates were analysed by western blotting for the phosphorylation of Ser 20 or Ser 46 with phospho-specific antibodies and for the occurrence of p53 as shown. b, Invitro-translated [35S]methionine-labelled Flag-tagged wild-type (WT) HIPK2, HIPK2K221A and HIPK2∆C proteins were immunoprecipitated with anti-Flag antibodies and either analysed by SDS-PAGE (left panels) or tested for their kinase activity in the presence of [γ-32P]ATP by kinase assays in vitro with GST–p53 as substrate (right panels). The kinase reaction was analysed by SDS–PAGE and the gel was exposed to two X-ray films, thus protecting the second film from the 35S signal. Autoradiograms (top and middle panels) and the Coomassie-stained gels (bottom

panels) are shown. c, H1299 cells were transiently transfected with p21Waf1 conjugated with the luciferase reporter gene, together with the indicated combinations of p53 (10 ng), p53-S46A (10 ng), HIPK2 (3 µg) and HIPK2-K221A (3 µg) and analysed for luciferase activity. Results are means ± s.d. for two independent experiments performed in duplicate. d, MCF-7 cells were irradiated with γ-rays (30 Gy) or UV (30 J m−2); 6 h later, endogenous HIPK2 was immunoprecipitated from cell lysates and its activity was determined by immune complex kinase assays (KA) with MBP as substrate. An autoradiogram from a reducing SDS gel shows phosphorylation of HIPK2 and MBP. e, MCF-7 cells were irradiated with UV (30 J m−2) for the indicated durations. Aliquots of cell lysates were tested for HIPK2 kinase activity (upper panels). Equal amounts of p53 were tested for Ser 46 phosphorylation (lower panels); representative experiments are shown.

approaches26–29,36, pointing to an important role for the PML-NB in p53 activation. However, it is still not clear whether PML-NBs are actually the sites of ongoing p53 target gene transcription. PMLNBs are found in the interchromosomal space37, arguing against active transcription. In contrast, the periphery of PML-NBs is known to contain nascent RNA38, but it remains to be determined whether this is really mRNA. Some PML-NB proteins such as CBP can rapidly move into and out of PML bodies39. It is therefore quite possible that PML-NBs constitute the sites for the post-translational modification of p53 and the assembly of active transcription factor complexes, which are then released from the PML-NBs to target their specific promoters. Although other kinases such as DLK are also contained in nuclear bodies40, this study identifies HIPK2 as the first protein kinase recruited to the PML-NBs. The specificity of PML3-mediated

recruitment of HIPK2 into PML-NBs was seen in further experiments in which PML3 failed to induce PML-NB localization of DLK (results not shown). Previous studies implicated the murine form of HIPK2 as a corepressor for homeodomain transcription factors31, suggesting that HIPK2 might modulate the function of several transcription factors serving as a corepressor or coactivator. The concept that different HIPK2 fractions bind to various target proteins is also compatible with the finding that only part of the pool of HIPK2 proteins is bound to p53. Here we show that human HIPK2 selectively phosphorylates p53 on Ser 46 both in vitro and in vivo. Our study reveals HIPK2 activation only in response to irradiation with UV, whereas it is known that Ser 46 is also phosphorylated by other p53-activating stimuli including γ-irradiation20, a stimulus that fails to trigger HIPK2 activity. This implies that HIPK2 is not the

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Figure 6 Physical and functional interactions between HIPK2 and CBP. a, Endogenous HIPK2 was immunoprecipitated (IP) from an aliquot of U2OS or MCF-7 cell lysates. Immunoprecipitated proteins were analysed by western blotting (WB) for the elution of CBP (upper panels) or HIPK2 (lower panels). b, HEK 293 cells were transfected with vectors encoding the indicated Flag-tagged HIPK2 variants and CBP (4 µg, respectively) as shown. Aliquots of cell lysates were precipitated with anti-CBP, anti-Flag or isotype-matched control antibodies, respectively. Co-precipitating HIPK2 was detected by immunoblotting. A non-specific band is marked with an asterisk. c, H1299 cells transfected to express the indicated combinations of expression vectors encoding p53 (1 µg), p53-K382R (1 µg), CBP (1 µg), HIPK2 (6 µg) and HIPK2-K221A (6 µg) were lysed and p53 was immunoprecipitated. The precipitate was analysed by immunoblotting for p53 acetylation by using an antibody recognizing p53 Lys 373/382 acetylation, and for p53 expression (see

accompanying manuscript by d’Orazzi et al.), an aliquot of the whole cell lysate was analysed for expression of CBP and HIPK2. The slower migration of HIPK2 than HIPK2-K221A is due to autophosphorylation. d, H1299 cells expressing the given combinations of p53 (1 µg), p53-S46A (1 µg), CBP (1 µg), HIPK2 (6 µg) and HIPK2K221A (6 µg) were analysed for protein expression and p53 Lys 373/382 acetylation as in c. e, H1299 cells were transfected with the pG13-Luc reporter gene together with the indicated combinations of expression vectors and tested for luciferase activity. Results are means ± s.d. for three independent experiments. f, U2OS cells transfected with the vector control, HIPK2 or HIPK2-K221A (8 µg of each) were either left untreated or irradiated with UV (30 J m−2) for 6 h. After immunoprecipitation with anti-p53 antibodies, extracts normalized for p53 amounts were tested for p53 Lys 373/382 acetylation (upper panel) and p53 (middle panel) and HIPK2 (bottom panel) levels by immunoblotting.

only kinase mediating the phosphorylation of p53 at Ser 46, but the identity and the activation signals for another p53 Ser 46 kinase remain to be revealed. One candidate is p38 mitogen-activated protein kinase19, but the importance of p38 in Ser 46 phosphorylation is not yet clear20,41. UV-induced p53 activation and apoptosis is concomitant with the phosphorylation of Ser 6, Ser 9, Ser 15, Ser 20,

Ser 37 and Ser 46 (refs 42, 43). The apoptosis-inducing function of p53 is impaired after the mutation of Ser 46 (ref. 20), showing the importance of this site for apoptosis. Our results indicate that modification of p53 by phosphorylation at Ser 46 and acetylation at Lys 382 mediated by the expression of human HIPK2 alone are not sufficient for the induction of p53-dependent apoptosis and instead



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Figure 7 Kinase-dependent cell proliferation block by HIPK2 expression. a, U2OS cells were transfected with a lac Z expression vector (100 ng) and 8 µg of plasmids for HIPK2, HIPK2-K221A or empty control vector. To ensure comparable transfection efficiencies, a sample of the cells was tested for β-galactosidase activity. Transfected cells were selected with G418 and surviving cell colonies were stained with crystal violet. Colony formation by the control vector was set as 100%; results are mean ± s.d. for three independent experiments (lower panel). b, The same experiment was performed on H1299 cells and evaluated as in a. c, U2OS cells were transfected with vectors encoding HIPK2 (2 µg) or HIPK2-K221A (2 µg). After 2 d, cells were stained and analysed by immunofluorescence. Single cells stained for DNA (DAPI), HIPK2 and BrdU are indicated by arrowheads; 200 triple-stained cells were analysed statistically (upper panel). d, The same experiment was performed with H1299 cells and evaluated as in c. e, U2OS cells transfected to express p53 (1 µg), HIPK2 (3 µg) or both were analysed by immunoblotting for the expression of p21Waf1 and β-tubulin.

cause cell-cycle arrest. It is therefore reasonable to assume that Ser 46 phosphorylation needs to occur in concert with the phosphorylation of further serines such as Ser 15 and Ser 20 to induce the expression of p53-dependent apoptosis genes. A role of HIPK2 in cell killing is revealed when these additional phosphorylation signals are provided by a sublethal dose of UV; this is in accordance 8

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Figure 8 HIPK2 participates in UV-mediated apoptosis. a, U2OS cells were transfected to express wild-type or kinase-deficient HIPK2 (8 µg). Cells were either left untreated (filled columns) or irradiated with a sublethal UV dose (10 J m−2; open columns). Apoptosis was quantified 24 h later by trypan blue exclusion; results are means ± s.d. b, U2OS cells were treated with either control morpholino oligonucleotides (15 µM) or HIPK2 antisense morpholino oligonucleotides (15 µM) for 48 h. A fraction of the cells was lysed and tested by western blotting (WB) for HIPK2 and β-actin expression. c, U2OS cells treated with control and HIPK2 antisense morpholino oligonucleotides were left untreated or irradiated with UV (50 J m−2) to induce apoptosis and analysed 8 h later. Apoptotic cells appear dark and round in these phase-contrast micrographs. Apoptosis was quantified by trypan blue exclusion (upper panels). d, A fraction of U2OS cells from the experiment in c was lysed and tested by western blotting for p53 Ser 46 phosphorylation and p53 expression.

with the data by Oda et al.20 showing the special relevance of Ser 46 to the induction of apoptosis. Death of UV-irradiated cells is enhanced by the overexpression of human HIPK2 and is impaired when lower levels of endogenous HIPK2 are present, thus revealing an important role for HIPK2 for this process. Our results indicate that HIPK2-mediated phosphorylation of p53 at Ser 46 is required for the CBP-mediated acetylation of p53 at Lys 382. In vitro data show that CBP/p300 binds to the N terminus of p53 (ref. 12), raising the possibility that the phosphorylation of Ser 46 facilitates the binding of CBP to its substrate p53. A similar situation occurs after the phosphorylation of Ser 15, which enhances p53 binding to CBP/p300 in vitro and in vivo12,24. In contrast to Ser 46, phosphorylation of Ser 15 is not required for p53 acetylation. A p53 protein mutated at Ser 46 can still be activated by HIPK2, albeit to a significantly smaller extent than the wild-type p53 protein. Although the mutant p53 protein is not directly phosphorylated by HIPK2 and acetylated by CBP, preliminary results indicate that HIPK2 also exerts indirect effects on p53 (T.G.H. and M.L.S., manuscript in preparation). Further studies will be required to elucidate a possible involvement of HIPK2 in genotoxic stress, DNA repair and tumour suppression. NATURE CELL BIOLOGY VOL 4 JANUARY 2002 http://cellbio.nature.com


articles Methods Cell culture and transfections HEK 293, SaOS-2, H1299, U2OS, HEp2 and MCF-7 cells were maintained in DMEM containing 10% FCS and 1% (w/v) penicillin/streptomycin and grown at 37 °C and 5% CO2. Cells were transiently transfected with the LipofectAMINE® (Gibco BRL), Effectene® or SuperFect® reagent (Qiagen Inc.) in accordance with the instructions of the manufacturers.

Antibodies and plasmids Antibodies recognizing Flag (M2), p21Waf1 (F-5), p53 (DO-1 and FL393) CBP (A22 and C-1) β-tubulin (H-239), PML (PG-M3) were from Santa Cruz Inc. and anti-acetyl-p53 antibodies from Upstate Biotechnology. The mouse monoclonal anti-phospho-p53-Ser 15 (16G8) and the rabbit polyclonal anti-phospho-p53-Ser 6, -Ser 9 and -Ser 20 antibodies were purchased from New England Biolabs. The anti-phospho-p53-Ser 46 (ref. 20) and rat anti-PML antibodies44 were as described previously. The affinity-purified rabbit polyclonal anti-HIPK2 antibody was generated by Eurogentech (Seraing, Belgium) and was raised against the following keyhole limpet hemocyanine (KLH)-coupled [peptide contained in human HIPK2: H2N-SSPQRSKRVKENTPPRC-COOH. The reporter plasmids pG13-Luc, pMG15-Luc45, p21Waf1-Luc35, Bax-Luc27 and the vectors encoding CBP46, PML-L and PML3 (ref. 27), HIPK2 (ref. 30), RSV-lacZ47 and bacterial expression vectors encoding the GST protein fused to p53 (refs 25, 27) and CBP46 were as published. Expression vectors for wild-type p53 and p53-K382A were gifts from S. Saito; A. J. Fornace, Jr, kindly provided the GST–p53-S46A expression vector. HIPK2 mutants and the GFP fusion protein were constructed by standard PCR techniques. The pCMV–Flag–p53-S46A construct was generated by cloning the EcoRI/BamHI fragment of GST–p53S46A into the vector pCMV–Tag-2B (Stratagene). All constructs were characterized by restriction digest and DNA sequencing.

GST pull-down assays The GST fusion proteins were expressed in Escherichia coli BL21pLysS and purified as described elsewhere48. HIPK2 proteins were labelled with [35S]methionine by using the coupled TnT in vitro transcription–translation system from rabbit reticulocyte lysates in accordance with the instructions of the manufacturer (Promega Inc.). Labelled proteins were incubated with 4 µg GST fusion protein at 4 °C for 3 h with in vitro interaction buffer (IVB) (20 mM Tris/HCl pH 8.0, 10% (v/v) glycerol, 0.2% (v/v) NP-40, 0.5 mM EDTA, 1 mM dithiothreitol, 200 mM NaCl) and washed three times in 750 µl IVB. The bound proteins were eluted with 1 × SDS sample buffer, separated by denaturing SDS–PAGE and analysed by autoradiography.

Luciferase reporter assays Cells were seeded in six-well plates and transfected with a constant amount of DNA consisting of 750 ng luciferase reporter construct, 250 ng RSV–lacZ expression vector and the indicated expression constructs. At 24–36 h after transfection, cell extracts were analysed for luciferase activity and results were normalized to the activity of β-galactosidase.

Immunofluorescence Cells were grown in 12-well plates on coverslips and either left untransfected or transfected with 10–300 ng of expression vector, washed once with PBS and fixed for 5 min at −20 °C with methanol/acetone (1:1). After being dried, cells were blocked for 30 min at 22 °C in PBS containing 5% (v/v) goat serum. Cells were then incubated for 60 min with the primary antibodies at 22 °C and washed five times (5 min each) in PBS before incubation for 45 min with appropriate fluorochromeconjugated secondary antibodies. The following secondary antibodies were used: Alexa-488-coupled goat anti-mouse and Alexa-488-coupled goat anti-rabbit (Molecular Probes), Cy3-coupled goat antirabbit, Texas red-coupled goat anti-mouse, amino-methylcoumarin (AMCA)-coupled goat anti-rat and AMCA-coupled goat anti-rabbit antibodies (Jackson Laboratories). In some experiments, chromosomal DNA was revealed by staining with 4′,6-diamidino-2-phenylindole (DAPI). Stained cells were mounted on glass slides and examined with an epifluorescence microscope (Axioplan-2; Zeiss) or a confocal laser microscope (Leica).

BrdU incorporation assays Cells grown on coverslips were fixed 36 h after transfection. BrdU incorporation was measured with the in situ cell proliferation kit FLUOS (Roche Molecular Biochemicals) in accordance with the manufacturer’s instructions. Images were acquired and analysed with an epifluorescence microscope (Axioplan-2).

Co-immunoprecipitation and western blotting Co-immunoprecipitation of endogenous proteins was performed after the lysis of (2–3) × 107 cells in high-salt lysis buffer (50 mM Tris/HCl pH 7.5, 10% (v/v) glycerol, 1% (v/v) NP-40, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM dithiothreitol, 10 mM NaF, 0.5 mM sodium vanadate, 10 µg ml−1 leupeptin, 10 µg ml−1 aprotinin). After centrifugation, equal amounts of protein contained in the supernatant were diluted with lysis buffer lacking NaCl and KCl to a final concentration of 100 mM NaCl and 50 mM KCl. After the lysates had been precleared with 5 µg control antibody and ProteinA/G–Sepharose beads, 5 µg affinity purified rabbit anti-HIPK2 antibodies and Protein-A/G–Sepharose beads were added and incubated overnight at 4 °C on a rotating wheel. The beads were washed three times in ice cold PBS containing 10 mM NaF and 0.5 mM sodium vanadate. Proteins bound to the control antibodies and to the anti-HIPK2 antibodies were eluted by boiling for 3 min in 1 × SDS sample buffer and analysed by reducing SDS–PAGE. For mapping interaction domains, HEK 293 cells were grown on 10-cm dishes and transiently transfected with 4 µg of the indicated constructs; 36 h later, cells were lysed and further processed essentially as described47.

Irradiation with UV and γ-rays, and in vitro kinase assays Cells were grown on 6-cm dishes, culture medium was removed and cells were irradiated with the indicated dose of UV with a StrataLinker (Stratagene). Subsequently, the culture medium was added

back and cells were further incubated for the indicated periods. A Gammacell 1000 radiator was used as the source for γ-irradiation. In vitro kinase assays were performed after the lysis of cell pellets in TOTEX buffer47. After centrifugation, supernatants were further analysed either by western blotting or immunoprecipitation after being precleared by the addition of 2 µg of anti-Flag or anti-HIPK2 antibodies and 25 µl Protein-A/G–Sepharose (Santa Cruz Inc.). After rotation for 4 h on a spinning wheel at 4 °C, the immunoprecipitates were washed three times in TOTEX buffer and twice in kinase buffer (20 mM Hepes/KOH pH 7.4, 25 mM β-glycerophosphate, 2 mM dithiothreitol, 20 mM MgCl2). The kinase assay was performed in a final volume of 30 µl kinase buffer containing 40 µM ATP, 5 µCi [γ32 P]ATP and 3 µg substrate protein. After incubation for 20 min at 30 °C, the reaction was stopped by the addition of 5 × SDS loading buffer. After separation by SDS–PAGE, the gel was fixed, dried and exposed to an X-ray film.

Knock-down experiments with antisense oligonucleotides The knock-down of endogenous HIPK2 expression was done with antisense morpholino oligonucleotides (GeneTools LLC). The HIPK2 antisense morpholino oligonucleotide 5′-CGCGGTTCATGGCAACGGAGAAGGG-3′ was used. As a control, a random morpholino oligonucleotide with the sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′ was used. The HIPK2 or control antisense morpholino oligonucleotides (15 µM final concentration) were added to the culture medium and U2OS cells were loaded by the scrape loading technique as described by the manufacturer; 2 d later, aliquots of the treated cells were analysed by western blotting for protein expression. For functional analysis, cells were irradiated and analysed as described in the figure legend.

Apoptosis assays Apoptosis was assessed by identifying dead cells by Trypan Blue exclusion as described48. In brief, 300 cells per experiment were analysed under a light microscope for unstained (live) or blue stained (dead) cells. RECEIVED 1 FEBRUARY 2001; REVISED 10 AUGUST 2001; ACCEPTED 19 SEPTEMBER 2001; PUBLISHED 10 DECEMBER 2001.

1. Momand, J., Zambetti, G. P., Olson, D. C., George, D. & Levine, A. J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237–1245 (1992). 2. Oliner, J. D. et al. Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 362, 857–860 (1993). 3. Haupt, Y., Maya, R., Kazaz, A. & Oren, M. Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299 (1997). 4. Kubbutat, M. H., Jones, S. N. & Vousden, K. H. Regulation of p53 stability by Mdm2. Nature 387, 299–303 (1997). 5. Oren, M. Regulation of the p53 tumor suppressor protein. J. Biol. Chem. 274, 36031–36034 (1999). 6. Yu, J. et al. Identification and classification of p53-regulated genes. Proc. Natl Acad. Sci. USA 96, 14517–14522 (1999). 7. Zhao, R. et al. Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev. 14, 981–993 (2000). 8. Ashcroft, M., Taya, Y. & Vousden, K. H. Stress signals utilize multiple pathways to stabilize p53. Mol. Cell. Biol. 20, 3224–3233 (2000). 9. Shieh, S. Y., Ikeda, M., Taya, Y. & Prives, C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334 (1997). 10. Unger, T. et al. Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2. EMBO J. 18, 1805–1814 (1999). 11. Fuchs, S. Y., Fried, V. A. & Ronai, Z. Stress-activated kinases regulate protein stability. Oncogene 17, 1483–1490 (1998). 12. Appella, E. & Anderson, C. W. Post-translational modifications and activation of p53 by genotoxic stresses. Eur. J. Biochem. 268, 2764–2772 (2001). 13. Hirao, A. et al. DNA damage-induced activation of p53 by the checkpoint kinase Chk2 Science 287, 1824–1827 (2000). 14. Shieh, S. Y., Ahn, J., Tamai, K., Taya, Y. & Prives, C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289–300 (2000). 15. Lakin, N. D., Hann, B. C. & Jackson, S. P. The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53. Oncogene 18, 3989–3995 (1999). 16. Burma, S. et al. DNA-dependent protein kinase-independent activation of p53 in response to DNA damage. J. Biol. Chem. 274, 17139–17143 (1999). 17. Nakagawa, K., Taya, Y., Tamai, K. & Yamaizumi, M. Requirement of ATM in phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks. Mol. Cell. Biol. 19, 2828–2834 (1999). 18. de Stanchina, E. et al. E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev. 12, 2434–2442 (1998). 19. Bulavin, D. V. et al. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J. 18, 6845–6854 (1999). 20. Oda, K. et al. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser46-phosphorylated p53. Cell 102, 849–862 (2000). 21. Liu, L. et al. p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol. Cell. Biol. 19, 1202–1209 (1999). 22. Ito, A. et al. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO. J. 20, 1331–1340 (2001). 23. Gu, W. & Roeder, R. G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606 (1997). 24. Sakaguchi et al. DNA damage activates p53 through a phosphorylation–acetylation cascade. Genes Dev. 12, 2831–2841 (1998). 25. Luo, J., Su, F., Chen, D., Shiloh, A. & Gu, W. Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 408, 377–381 (2000).



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ACKNOWLEDGEMENTS We are grateful to S. Soddu for sharing results before publication, E. Appella and L. Florin for helpful comments on the manuscript, T. Hamid and N. Stephan for excellent technical assistance, P. Gutwein for help with microscopy, and all colleagues who generously provided plasmids and reagents: W. Gu, G. Haegeman, M. Oren, B. Vogelstein, G. del Sal, G. Blandino and A. J. Fornace Jr. Our laboratories are supported by grants from the EU (QLK3-CT-2000-00463), Deutsche Krebshilfe, Deutsche Forschungsgemeinschaft (Schm 1417/3-1), Universität Heidelberg and Fonds der chemischen Industrie. The HPI is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit. Correspondence and requests for materials should be addressed to M.L.S.



supplementary infomation

Figure S1 HIPK2-mediated phosphorylation of p53. HIPK2 and HIPK2-K221A were expressed in HEK^293 cells, isolated by immunoprecipitation and used for immune complex kinase assays using the indicated GST–p53 fusion proteins as substrates. a, The full length p53 protein fused to GST was used as a substrate. b, GST–p53(1-80) was used as a substrate. c, GST–p53(1-80) or a point mutant of this fusion protein containing a Ser 46 to alanine exchange were employed as substrate proteins, typical autoradiograms are displayed.

Figure S2 UV-radiation does not alter endogenous HIPK2 levels. MCF-7 cells were treated with UV-radiation as indicated and incubated for another 6^h. HIPK2 immunoprecipitates were analysed by western blotting for the occurrence of HIPK2.

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