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Oncogene (2003) 22, 8731–8737 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Sp100 is important for the stimulatory effect of homeodomain-interacting protein kinase-2 on p53-dependent gene expression Andreas Möller1,2, Hüseyin Sirma3, Thomas G Hofmann3, Hannah Staege3, Ekaterina Gresko2, Katharina Schmid Lüdi2, Elisabeth Klimczak1, Wulf Dröge1, Hans Will3 and M Lienhard Schmitz*,2 1 German Cancer Research Center, Division of Immunochemistry (G0200), Im Neuenheimer Feld 280, Heidelberg D-69120, Germany; University of Bern, Department of Chemistry and Biochemistry, University of Bern, Freiestr. 3, Bern 3012, Switzerland; 3Department of General Virology, Heinrich-Pette-Institute for Experimental Virology and Immunology, Martinistrasse 52, D-20251 Hamburg D-20251, Germany 2 HIPK2 shows overlapping localization with p53 in promyelocytic leukemia (PML) nuclear bodies (PMLNBs) and functionally interacts with p53 to increase gene expression. Here we demonstrate that HIPK2 and the PML-NB resident protein Sp100 synergize for the activation of p53-dependent gene expression. Sp100 and HIPK2 interact and partially colocalize in PML-NBs. The cooperation of HIPK2 and Sp100 for the induction of p21Waf1 is completely dependent on the presence of p53 and the kinase function of HIPK2. Downregulation of Sp100 levels by expression of siRNA does not interfere with p53mediated transcription, but obviates the enhancing effect of HIPK2. In summary, these experiments reveal a novel function for Sp100 as a coactivator for HIPK2-mediated p53 activation. Oncogene (2003) 22, 8731–8737. doi:10.1038/sj.onc.1207079 Keywords: HIPK2; Sp100; p53; PML nuclear body Introduction The nucleus is highly organized into discrete substructures including the promyelocytic leukemia nuclear bodies (PML-NBs) (Salomoni and Pandolfi, 2002). The physiological functions of PML-NBs are still controversially discussed and the proposed relevance for transcriptional regulation (Eskiw and Bazett-Jones, 2002), replication (Sourvinos and Everett, 2002) and genomic stability (Zong et al., 2000) or as a nuclear depot (Negorev and Maul, 2001) is reviewed elsewhere (Borden, 2002). Interestingly, PML-NBs are frequently targeted by viral infections and disrupted in acute promyelocytic leukemia (Weis et al., 1994). Among the permanent residents of PML-NBs are several critical regulators of cell proliferation, apoptosis and genome *Correspondence: M Lienhard Schmitz; E-mail: [email protected] Received 2 May 2003; revised 2 August 2003; accepted 5 August 2003 stability, including HAUSP(USP7), Daxx, pRB, BLM and Sp100 (Seeler and Dejean, 2001). The Sp100 protein was identified using the sera from patients suffering from primary biliary cirrhosis and, due to its ‘speckled’ appearance in the nucleus, termed Sp100 (for speckled protein of 100 kDa) (Szostecki et al., 1990). The Sp100 family of proteins comprises the Sp110, Sp140/LYSp100 and the autoimmune regulator protein (AIRE), a transactivator which is mutated in a hereditary autoimmune disease (Vogel et al., 2002). All members of the Sp100 protein family share an N-terminal HSR (homogeneously staining region) domain (Sternsdorf et al., 1999). Sp100 proteins occur in at least four different spliced forms, with Sp100 A (480 amino acids, migrating aberrantly at 90–100 kDa) being the most abundant form (Rogalla et al., 2000; Negorev and Maul, 2001; Seeler and Dejean, 2001). The larger variant Sp100 HMG encodes additional functional domains such as a SAND domain and a HMG box. Both splice variants can be covalently modified by SUMO-1 and are transcriptionally upregulated by interferons (Guldner et al., 1992). When attached to DNA upon fusion to the DNA-binding domain of the yeast Gal4 protein, Sp100 inhibits basal transcription. The Sp100 protein bears a C-terminal transactivation domain that is active in the full-length protein in yeast, but not in mammalian cells where it is masked by the Nterminus (Bloch et al., 2000). Sp100 most likely does not bind to DNA alone, but may be recruited to the DNA via association with DNA-binding proteins such as hHMG2/DSP1, heterochromatin protein 1 (HP1) (Seeler et al., 1998), the B-cell-specific transactivator Bright (Zong et al., 2000) or ETS-1 (Wasylyk et al., 2002). While interaction of Sp100 with hHMG2/DSP1, HP1 or Bright mediates transcriptional repression, binding to ETS-1 stimulates the expression of ETS-1 target genes. Among the proteins inducibly associating with a subfraction of PML-NBs are the serine/threonine kinase HIPK2, the transcriptional regulator p53 and the acetylase CBP. HIPK2 and its homolog HIPK1 can bind via their C-terminal sequences to p53 (D’Orazi et al., 2002; Hofmann et al., 2002; Kim et al., 2002; Kondo et al., 2003). Binding of HIPK2 to p53 leads to Sp100 is important for stimulatory effect A Möller et al 8732 Results Sp100 and HIPK2 cooperate to induce p53-dependent gene expression To investigate the impact of Sp100 on HIPK2-induced p53 activity, human U2OS cells were transfected with various combinations of expression vectors encoding Sp100 and the wild-type and kinase inactive (K221A) point mutant of HIPK2, along with a pG13-luc luciferase reporter gene controlled by multimers of intact p53-binding sites (Figure 1a). While expression of HIPK2 induced p53-dependent transcription, Sp100 or kinase inactive HIPK2 failed to trigger reporter gene expression. Coexpression of HIPK2 and Sp100 synergistically stimulated reporter gene activity, thus suggesting a transactivating role for Sp100. To ensure that the observed effects are dependent on the p53-binding sites, the experiment was repeated using the pMG15-luciferase reporter gene containing mutated p53-binding sites in the promoter (Figure 1b). The absence of intact p53 sites precluded HIPK2/Sp100-mediated gene induction, thus revealing that the observed effects are p53dependent. To measure the impact of Sp100 on HIPK2-mediated gene expression in the context of a natural p53dependent promoter, p53-deficient H1299 cells were transfected with a p21Waf1 promoter fused to the luciferase gene together with vectors encoding HIPK2, p53 and Sp100 (Figure 2a). In the absence of p53, coexpression of HIPK2 and Sp100 failed to induce transcription of p21Waf1. Upon expression of p53, HIPK2-mediated upregulation of p21Waf1 was further boosted by Sp100. To address the question as to whether HIPK2 and Sp100 also synergize for the induction of p21Waf1 expression within its natural chromatin context, U2OS cells were transfected with vectors encoding Oncogene a Luciferase fold activation pG13-luc 15 10 5 HIPK2 WT - + HIPK2 K221A - - Sp100 - - b - + - + - - + - + + + - pMG15-luc Luciferase fold activation phosphorylation of p53 at serine 46, thus enabling subsequent CBP-mediated acetylation of p53, which in turn promotes p53-dependent target gene expression. Activated p53 then mediates either growth arrest at the G1/S or G2/M cell cycle transitions or apoptosis (Hofmann et al., 2002). Accordingly, the stimulatory function of HIPK2 on p53 enhances the expression of p53 target genes such as p21Waf1 or Bax and stops cell proliferation or induces apoptosis depending on its kinase activity (Greenwood, 2002). Immunofluorescence studies revealed a minor fraction of HIPK2 in PMLNBs (Möller et al., 2003). Treatment of cells with UV irradiation and As2O3 increased the colocalization between p53 and HIPK2 in PML-NBs, while expression of the PML isoform PML-IV efficiently translocated HIPK2 into this subnuclear structure (D’Orazi et al., 2002; Hofmann et al., 2002). Given the corecruitment of HIPK2 and p53 to PML-NBs, we analysed the effects of the putative transcriptional regulatory Sp100 protein on HIPK2/p53-mediated transcription. A variety of experimental approaches revealed Sp100 as a coactivator for HIPK2/p53-mediated transcription. 15 10 5 HIPK2 WT - + - - + - HIPK2 K221A - - + - - + Sp100 - - - + + + Figure 1 Synergistic activation of p53-dependent gene expression by HIPK2 and Sp100. (a) U2OS cells were cotransfected with luciferase reporter constructs controlled by multimers of intact (pG13-Luc) p53-binding sites and expression vectors encoding HIPK2, HIPK2 K221A or Sp100 as shown. (b) The experiment was done as in (a), with the exception that a reporter gene controlled by multimers of mutated (pMG15-Luc) p53-binding sites was used. At 1 day post-transfection, cells were harvested and tested for luciferase activity. Transactivation by the empty expression vector was arbitrarily set as 1; bars indicate standard deviations from three independent experiments HIPK2 and/or Sp100 and analysed for p21Waf1 by Western blotting. While HIPK2 alone increased p21Waf1 protein levels, coexpression of Sp100 led to a further increase in the amount of this cell cycle regulator (Figure 2b). The transcriptional synergism between HIPK2 and Sp100 for induction of the p21Waf1 promoter also occurred in nontransformed HS27 cells (Figure 2c), thus ensuring the physiological relevance of the observed effects. Mapping of Sp100 and HIPK2 domains mediating the cooperative effect on p53-induced transcription In order to map the HIPK2 region mediating this cooperative effect with Sp100, various deletion mutants of HIPK2 were tested in the absence or presence of coexpressed Sp100 for their effect on the p21Waf1 promoter-dependent luciferase reporter gene (Figure 3). HIPK2 variants lacking either the C-terminal or N-terminal regions or consisting of the kinase domain all failed to cooperate with Sp100 for the induction of Sp100 is important for stimulatory effect A Möller et al 8733 a Luciferase fold activation Luciferase fold activation 20 10 p53 HIPK2 WT HIPK2 K221A - - - - - - - + - - - + + + + + + 10 + - Sp100 HIPK2 WT - - + - - + - - + HIPK2 K221A - + - - - + - - - + - HIPK2 ∆C - - - - HIPK2 ∆N HIPK2 KD - - - - - - - + + + α p21 Waf1 α FLAG 20 + - - - + + + HIPK2 Sp100 30 - - + - - + - Sp100 b p21Waf1-luc p21Waf1-luc - - + - + + + HIPK2 - + + + + + - - - - + - - - + - + - - - + - - - + - - - + - - - - - - - + + Figure 3 Mapping of the HIPK2 domains mediating cooperation with Sp100. A p21Waf1-luciferase reporter gene and the indicated HIPK2 variants were expressed either alone or together with Sp100 in U2OS cells. The next day, extracts were prepared and tested for luciferase activity; results are displayed as in Figure 2. Correct expression of the FLAG-tagged proteins was ensured by Western blotting (data not shown). Data are from two experiments performed in duplicate; bars indicate standard deviations Sp100 α β-Actin p21Waf1-luc Luciferase fold induction c 6 4 2 pcDNA3 + - - - HIPK2 WT - + - + Sp100 - - + + Waf1 Figure 2 Sp100 and HIPK2 cooperate to induce p21 expression. (a) H1299 cells were transfected with a p21Waf1-luciferase reporter gene in combination with expression vector for p53, HIPK2, HIPK2 K221A or Sp100 as shown. Reporter gene activation induced by empty control vector was arbitrarily set as 1. Error bars represent standard deviations from three independent experiments performed in duplicate. (b) U2OS cells were transfected with the indicated expression vectors encoding FLAGtagged HIPK2 or FLAG-tagged Sp100. Aliquots of cell lysates were tested by Western blotting for p21Waf1 (upper) and expression of HIPK2, Sp100 and the loading control b-actin (lower). (c) HS27 cells were transfected with a p21Waf1-luciferase reporter gene and the indicated expression vectors for HIPK2 and Sp100. Results from two independent experiments performed in triplicate are shown; error bars indicate the standard deviations p21Waf1, indicating that the entire kinase is required for this functional interaction. The relevance of various Sp100 domains allowing cooperative induction of gene expression was mapped using a similar experimental approach. Sp100 mutants lacking the indicated functional domains (Figure 4a) were expressed either alone or in combination with HIPK2 and the p21Waf1 luciferase reporter gene. Proteins lacking either the N-terminal HSR domain or the C-terminal sequences failed to significantly support HIPK2/p53-triggered transcriptional activation. Also, the Sp100 variant lacking the C-terminal transactivation domain (Sp100 1–334) did not enhance HIPK2-mediated transcription. A point mutant with a lysine-to-alanine exchange in the SUMOlation site (Sp100 K297R SUMO ) still fully retained its ability to synergize with HIPK2 for the activation of the p21Waf1 (Figure 4b), arguing against the relevance of SUMO modification for this synergism. HIPK2 and Sp100 interact and partially colocalize To investigate whether the functional interaction between Sp100 and HIPK2 also involves physical association of both proteins, coimmunoprecipitation experiments were performed. HIPK2 was immunoprecipitated from U2OS cell extracts. Subsequent immunoblotting revealed that Sp100 coimmunoprecipitated only with aHIPK2 antibodies, but not with control antibodies, demonstrating the interaction of both endogenous proteins in vivo (Figure 5a). Colocalization of both proteins was investigated by expressing green fluorescent protein (GFP)-tagged HIPK2 and Sp100 in cells. Oncogene Sp100 is important for stimulatory effect A Möller et al 8734 a Immunoprecipitation HSR TAD HSR TAD SAND HMG-Box Co nt ro l HI PK IgG 2 700 800 900 α HMG 600 e Sp100 400 500 SUMO MHC Homology A 300 α 100 200 Amino Acids Ly sa t a HMG-Box Sp100 Sp100 128-478 WB: α Sp100 IgGH Sp100 296-890 Sp100 339-890 Sp100 619-890 WB: α HIPK2 Sp100 704-890 HIPK2 Sp100 1-334 b K/A Sp100 SUMO b HIPK2 + Sp100 + Sp100 128-478 + Sp100 296-890 + Sp100 339-890 + Sp100 704-890 + Sp100 208-480 + Sp100 SUMO + Sp100 1-334 + Sp100 619-890 + -fold activation of p21Waf1-Luc 0 5 10 15 20 25 Figure 4 Mapping of the Sp100 domains mediating synergism with HIPK2. (a) Schematic representation of the Sp100 A and Sp100 HMG proteins and their derivatives. The positions of amino acids are given at the left side; the domain structures are indicated. (b) U2OS cells were transfected with a p21Waf1-luciferase gene and expression vectors for the indicated Sp100 variants and HIPK2 as shown. Reporter activity is given as fold induction; error bars show standard deviations derived from three independent experiments Immunofluorescence staining revealed predominant localization of HIPK2 in discrete subnuclear speckles which were partially colocalizing with the Sp100 protein (Figure 5b). Colocalization between the endogenous proteins was investigated in U2OS cells, which showed areas of overlapping residence for both proteins in nuclear bodies, as revealed by confocal microscopy (Figure 5c). Sp100 contributes to HIPK2-mediated p53 activation The importance of Sp100 for HIPK2-mediated p53 activation was investigated by testing the effect of siRNAs specific for Sp100 on HIPK2/p53-induced Oncogene GFP-HIPK2 Sp100 HIPK2 Sp100 Merge Hoechst Merge DraQ5 c Figure 5 Sp100 and HIPK2 co-precipitate and colocalize in the nucleus. (a) U2OS cells were treated for 8 h with INFg and lyzed. Endogenous HIPK2 was immunoprecipitated from an aliquot of the cell lysates with rabbit polyclonal aHIPK2 antibodies. Control antibodies were added to another aliquot of the cell extract. Proteins were eluted from the washed immunoprecipitates with 1 SDS sample buffer and analysed by Western blotting either for the occurrence of Sp100 (upper) or HIPK2 (lower). (b) U2OS cells were transfected to express GFP-HIPK2. GFP-HIPK2 was detected by the intrinsic fluorescence of GFP; Sp100 (red) was visualized by indirect immunofluorescence. An overlay of both stainings reveals areas of colocalization in yellow, as indicated by the arrows. Nuclear DNA was visualized with Hoechst. (c) U2OS cells were stained by indirect immunofluorescence for endogenous HIPK2 (green) and endogenous Sp100 (red), and analysed by confocal microscopy. Areas of overlapping localization (yellow) are indicated by arrows transcription. A control experiment ensured that transfection of U2OS cells with pSUPER-Sp100, a vector that directs the synthesis of small interfering RNAs (siRNAs) specific for Sp100, leads to a reduction of relative Sp100 expression levels (Figure 6a). The incomplete reduction can be attributed to the limited transfection efficiency. To look for the effects on gene expression, U2OS cells were transfected with the p21Waf1 luciferase reporter gene and expression vectors encoding HIPK2 in the absence or presence of pSUPER-Sp100. HIPK2-induced transcription was impaired in the presence of Sp100 siRNAs (Figure 6b), indicating that Sp100 is important for stimulatory effect A Möller et al 8735 a triggered transcription was decreased. These experiments suggest that Sp100 acts upstream from p53. WB: αSp100 Sp100 β-Actin β α β-Actin pSUPER + - pSUPER-Sp100 - + b p21Waf1-luc Luciferase fold activation 8 * 6 4 2 HIPK2 pSUPER - + - + + + - pSUPER-Sp100 - - - + c Luciferase Fold activation p21Waf1-luc * 10 5 - - - - - - + + + + + + - + - - + + - + - - + + pSUPER - - + - + - - - + - + - pSUPER-Sp100 - - - + - + - - - + - + p53 HIPK2 wt Figure 6 Interference with Sp100 expression attenuates HIPK2mediated activation of p53-dependent gene expression. (a) U2OS cells were transfected either with the empty pSUPER control vector or pSUPER-Sp100, which allows production of a siRNA attenuating endogenous Sp100 expression. Equal amounts of protein contained in lysates were tested by immunoblotting for the occurrence of Sp100 (upper) and b-actin (lower). (b) U2OS cells were transiently transfected with a p21Waf1-dependent reporter gene, along with an expression vector encoding HIPK2, the pSUPER vector or pSUPER-SP100, and luciferase activity was determined. (c) H1299 cells expressing the indicated combinations of p53 and HIPK2 were tested for p21Waf1-luciferase production in the absence (pSUPER) or presence (pSUPER-Sp100) of siRNA specific for Sp100. In all experiments, the mean values showing luciferase activity from four independent experiments performed in duplicate are displayed. Error bars show standard deviations; the statistical significance (Po0.01) is indicated by a star diminished amounts of Sp100 negatively interfere with HIPK2 function. In order to learn whether interference with Sp100 expression targets only HIPK2- or also p53mediated signals, H1299 cells were transfected with the p21Waf1 luciferase reporter gene along with various combinations of p53 and HIPK2 expression vectors in the absence or presence of pSUPER-Sp100 (Figure 6c). Reduced Sp100 expression remained without any impact on p53-induced gene expression, while HIPK2/p53- Discussion Here we show that Sp100 and HIPK2 cooperate to induce p53-dependent gene expression, as revealed by a variety of experimental approaches. Enhancement of transcriptional activity occurs in the absence of Sp100 DNA binding, as the coactivating function was also seen for Sp100 A, the predominant Sp100 variant that lacks the DNA-binding SAND and HMG domains. Although Sp100 contains no typical coactivator consensus motifs such as the LXXLL signature, the integrity of the C-terminal transactivation domain is important for its coactivating function. Also, the deletion of N-terminal sequences important for PML-NB recruitment and Sp100 self-aggregation (Negorev et al., 2001) precluded the coactivator function. Similar to the PML protein, Sp100 also displays transcriptional regulatory properties. Sp100 has been implicated in transcriptional repression (Lehming et al., 1998) and there is also recent evidence for a role as a coactivator for ETS-1driven transcription (Wasylyk et al., 2002). This work adds evidence for a role of Sp100 in transcriptional coactivation. HIPK2 and Sp100 interact, and partial colocalization is seen in distinct nuclear bodies and the nucleoplasm. It is currently not clear whether both proteins bind directly or indirectly via contact to a common binding partner. Since both proteins have several reported binding partners, the stoichiometry or the existence of distinctly composed complexes remains to be solved. We also investigated the possible HIPK2mediated phosphorylation of Sp100, but several in vivo and in vitro experiments failed to give evidence for such a direct phosphorylation under the conditions used (data not shown). The stimulatory role of Sp100 for HIPK2 function may be explained by several mechanisms: (I) HIPK2mediated events could cause a loss of Sp100-mediated repression, for example, by lowering the affinities of Sp100 to repressor proteins. Accordingly, HIPK2 can control the corepressor activity of Groucho (Choi et al., 1999). Since the interaction of Sp100 with members of the HP1 family of nonhistone chromosomal proteins is regulated by SUMOlation (Seeler et al., 1998), the full coactivator function of Sp100 SUMO mutant argues against an involvement of HP1 proteins. (II) HIPK2 could induce the oligomerization of Sp100 with the transactivating Sp100 family members Sp110 and Sp140/LYSp100 or other transcriptional activators. (III) The reported decomposition of PML-NBs mediated by HIPK2 (Engelhardt et al., 2003) or HIPK1 (Ecsedy et al., 2003) could possibly induce relocation of Sp100 to opened chromatin sites allowing transcriptional initiation. Since overexpressed Sp100 is preferentially deposited outside from PML-NBs (Negorev and Maul, 2001) and mutual binding of HIPK2 and Sp100 was only observed after interferon-induced upregulation of Sp100, it might be possible that HIPK2/Sp100 Oncogene Sp100 is important for stimulatory effect A Möller et al 8736 interactions occur outside from PML-NBs. Accordingly, the main fraction of HIPK2 is contained in HIPK domains that are distinct from PML-NBs (Möller et al., 2003). Further support for the idea that the coactivating function of Sp100 is exerted outside from the PML-NBs comes from the finding that the coactivating function of Sp100 for transcription factor ETS-1 occurs after ETS1-mediated displacement of Sp100 from PML-NBs (Wasylyk et al., 2002). (IV) Our experiments show that Sp100 is important for HIPK2-mediated effects, while p53-driven transcription remains unaffected. This selectivity shows that Sp100 acts upstream from p53, and may be taken as an indication that the function of Sp100 as a repressor or a coactivator might depend on the activating signal and/or the chromatin context. Immunofluorescence Cells were grown in 12-well plates on coverslips and either left untransfected or transfected with 100 ng of the indicated expression vectors. The next day, cells were washed with phosphate-buffered saline (PBS) and fixed for 1 min at 201C with methanol/acetone (1 : 1). After drying, cells were rehydrated in PBS and blocked in PBS containing 5% (v/v) goat serum for 30 min. Cells were then incubated with the primary antibodies for 60 min at room temperature and washed five times for 5 min in PBS before incubation with the appropriate fluorochrome-conjugated secondary antibodies for 45 min. The following secondary antibodies were used: Alexa Fluor488-coupled goat anti-rabbit and Alexa Fluor 594-coupled goat anti-rat (Molecular Probes). After washing once with PBS, chromosomal DNA was stained with Hoechst 33258 (1 mg/ml) or DraQ5 for 15 min. Cells were washed four more times in PBS, mounted on glass slides and examined using a confocal laser microscope (Leica). Materials and methods Co-precipitation experiments and immunoblotting Cell culture and transfections Human U2OS, HS27, and H1299 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin (all from Life Technologies). These cell lines were transfected using the Superfects reagent (Qiagen Inc.), according to the instructions of the manufacturer. Plasmids and antibodies The reporter plasmids pG13-Luc, pMG15-Luc (Kern et al., 1992) and p21Waf1-Luc (el Deiry et al., 1993), as well as the vectors encoding HIPK2, HIPK2 K221A, HIPK2DC (amino acids 1–520), HIPK2DN (amino acids 551–1191), HIPK2 KD (amino acids 189–520) and GFP-HIPK2 were published (Hofmann et al., 2002). Sp100 and its mutant derivatives were described (Sternsdorf et al., 1999). The pSUPER-Sp100 vector was constructed by inserting the annealed oligonucleotides: 5-GATCCCCACCAGTAGCAAATGAGATGTTCAA GAGACATCTCATTTGCTACTGGTTTTTTGGAAA and 5-AGCTTTTCCAAAAAACCA GTAGCAAATGAGATGT CTCTTGAACATCTCATTTGCTACTGGTGGG in the pSUPER vector (Brummelkamp et al., 2002) opened with BglII and HindIII. All constructs were characterized by restriction digest and DNA sequencing. Antibodies recognizing Flag (M2), p21Waf1 (F-5), p53 (DO-1) and b-actin were from Santa Cruz Inc. The rat aSp100 antibody (Sternsdorf et al., 1999) and affinity-purified rabbit polyclonal aHIPK2 antibody (Hofmann et al., 2002) were described. Luciferase assays Total cell extracts were measured in a luminometer (Duo Lumat LB 9507, Berthold) by automatically injecting 50 ml of assay buffer and measuring light emission for 10 s after injection according to the instructions of the manufacturer (Promega Inc.). Luciferase activities were normalized on the basis of b-galactosidase activity of a cotransfected RSV-b-gal vector. Cell extracts contained in NP-40 high salt lysis buffer (20 mM Tris/HCl pH 7.5, 300 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 10 mM NaF, 0.5 mM sodium vanadate, leupeptine (10 mg/ml), aprotinin (10 mg/ml), 1% (v/v) NP-40 and 10% (v/v) glycerol) were diluted with salt-free NP-40 buffer to a final NaCl concentration of 150 mM. Extracts were either directly analysed by Western blotting or immunoprecipitated following preclearance with protein A/G sepharose and 2 mg of a control IgG. A volume of 2 mg of precipitating antibodies and 25 ml of protein A/G sepharose were added to the precleared lysate and rotated 6 h on a spinning wheel at 41C. The immunoprecipitates were washed 5 in lysis buffer and eluted by boiling in 1 SDS sample buffer. Following separation by SDS–PAGE, proteins were blotted to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked and then incubated in TBST containing the primary antibody and 2% (w/v) milk powder. The respective proteins were incubated with an appropriate secondary antibody coupled to horseradish peroxidase, and visualized by enhanced chemiluminescence according to the instructions of the manufacturer (NEN). Acknowledgements Our work was supported by grants from the Deutsche Forschungsgemeinschaft (Schm 1417/3-1), Fonds der chemischen Industrie, EU project (QLK3-CT-2000-00463) sponsored by the Schweizerisches Bundesamt für Bildung und Wissenschaft, Oncosuisse, Schweizerischer Nationalfonds, Association for International Cancer Research, ‘Stiftung zur Förderung der wissenschaftlichen Forschung an der Universität Bern’ and a grant from the Roche Research Foundation awarded to AM and MLS. 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