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Cancer Research 67, 66, January 1, 2007. doi: 10.1158/0008-5472.CAN-06-1671
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Daxx Cooperates with the Axin/HIPK2/p53 Complex to Induce Cell Death

Qinxi Li1, Xuan Wang1, Xiaoling Wu1, Yanning Rui2, Wei Liu2, Jifeng Wang1, Xinghao Wang3, Yih-Cherng Liou4, Zhiyun Ye1 and Sheng-Cai Lin1,2

1 Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China; 2 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; 3 Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah; and 4 Department of Biological Science, National University of Singapore, Singapore

Requests for reprints: Sheng-Cai Lin, School of Life Sciences, Xiamen University, Fujian 361005, China. Phone: 86-592-218-2993; Fax: 86-592-218-2993; E-mail: linsc{at}xmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Daxx, a death domain–associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control ß-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not Daxx{Delta}Axin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser46 phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin–/– cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death. [Cancer Res 2007;67(1):66–74]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Daxx was initially identified as a protein that binds to the Fas death domain and has been implicated in a Fas-mediated apoptotic pathway by serving as an adaptor protein linking Fas signaling to c-Jun-NH2-kinase (JNK) pathways via apoptosis signal–regulating kinase 1 (1, 2). Genetic studies showed that mouse embryos deficient in Daxx cannot survive beyond embryonic days 8.5 to 9.5, indicating that Daxx is necessary in the early development of mouse embryos. However, results from TUNEL assays gave evidence that embryos with disrupted Daxx genes displayed extensive apoptosis, suggesting that Daxx may play a protective role in preventing apoptosis in the early embryo. Similarly, Daxx silencing by small interfering RNA (siRNA) was reported to sensitize cells to multiple apoptotic pathways, implying an antiapoptotic role for Daxx (3, 4).

Nevertheless, several lines of evidence have shown that Daxx may indeed exert proapoptotic functions, and that Daxx may play opposing roles with respect to apoptosis depending on the context (5–13). In Daxx–/– cells, the apoptosis rates in response to serum starvation were only slightly increased, which makes it difficult to conclude that Daxx deficiency–caused apoptosis in mutant mouse tissues is a direct consequence of a loss of antiapoptotic function of Daxx. In addition, it is clear that Daxx is required to potentiate stress-induced cell death in cell lines. It has been shown that Daxx induces apoptosis by interacting with several nuclear proteins, such as PML and HIPK2 in the nucleus (14, 15). It was also shown that Daxx interacted with p53 and promoted p53-dependent apoptosis (11). However, another study contradicted such an observation, showing that Daxx does not coimmunoprecipitate with wild-type p53, but only with tumorigenic mutant forms of p53. Intriguingly, only the Daxx-interacting mutants of p53 could inhibit stress-induced Daxx-mediated cell death (10). Although it remains unclear how Daxx modulates p53 function, the existing evidence clearly points to a functional linkage between Daxx and p53. A recent finding that Daxx interacts with, and inhibits, the transcriptional activity of Tcf4, which plays a critical role in maintaining the proliferative status of the stem cells in the crypts of the intestine (16, 17), suggests that Daxx may inhibit cell proliferation via multiple mechanisms.

Axin is a negative regulator of Axis formation in the development of mouse embryos; its deficiency leads to axis duplication (18). It acts as an architectural platform for the degradation of the oncogenic protein ß-catenin (19–23). Axin has, in fact, emerged as a major scaffold for many other pathways, including JNK mitogen-activated protein kinase signaling, p53 signaling, and transforming growth factor ß (TGF-ß) signaling (24–28). Recently, heterotrimeric G{alpha} subunits activated upon prostaglandin E2 stimulation were shown to interact with Axin, thereby disrupting the Axin/GSK3ß degradation complex and leading to stabilization of ß-catenin (22, 23). Most relevantly, we previously found that Axin forms a complex with p53 and its regulatory kinase HIPK2. Knockdown of Axin by siRNA reduced UV-induced p53 Ser46 phosphorylation and p53-mediated apoptosis (28). In addition, HIPK2 has been shown to interact with Daxx (15). All these observations prompted us to reevaluate a then seemingly unlikely clone identified by a yeast two-hybrid screen using full-length Axin (which encoded Daxx) as bait many years ago. Here, we show that Daxx interacts strongly with Axin both in vivo and in vitro, in that Axin serves as a scaffold for the assembly of the Axin/Daxx/HIPK2/p53 complex to promote the phosphorylation of p53 at Ser46 by HIPK2. The results provide an important mechanistic link for Daxx to tumor suppressors, p53 and Axin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids. The full-length cDNA encoding Daxx was amplified using cDNA generated from mRNA of HeLa cells using Pfu polymerase, and was inserted into pCMV5 expression vector after sequence verification. Deletion mutants were generated using standard techniques as previously described (28).

Preparation of antibodies. Mouse anti-HA (F-7), anti-Myc (9E10), anti-Hsp60 (H-1), anti-p53 (DO-1), and rabbit anti-p53 (FL393) antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Mouse anti-FLAG (M2) and anti-ß-actin were purchased from Sigma. Mouse anti–cytochrome c monoclonal antibody was a product of BD Biosciences. Rabbit anti–acetyl-p53-Lys320 and anti–acetyl-p53-Lys373 antibodies were purchased from Upstate Biotechnology, Inc. Anti-phospho-p53-Ser15, anti-phospho-p53-Ser20, anti-phospho-p53-Ser46, and anti-acetylated-p53-Lys382 rabbit antibodies were all purchased from Cell Signaling Technology (Sigma, Saint Louis, MO). The polyclonal antibody against Axin (C2b) has been previously described (28), and rabbit polyclonal antibody against Daxx was prepared by injecting the protein region of amino acids 625 to 740.

Cell culture, transient transfection, immunoprecipitation, and Western blotting. HEK293, HEK293T, H1299, U2OS, SaOS-2, HeLa, MCF-7, and SNU-475 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 IU of penicillin, and 100 mg/mL of streptomycin. Transient transfections were carried out using Dosper (Roche, Penzberg, Germany), LipofectAMINE 2000 (Invitrogen, Carlsbad, CA), or calcium phosphate precipitation method. Cell lysate preparation and immunoprecipitation were carried out as detailed previously (24). To determine whether Axin, Daxx, and p53 form a ternary complex, a two-step coimmunoprecipitation was done as previously described (28).

Immunokinase assay. H1299 cells were transfected with p53, Daxx, Axin, HIPK2, p300, or their mutants as indicated. At 30 h posttransfection, cells were harvested with lysis buffer. p53 was immunoprecipitated with anti-FLAG or anti-HA antibody; phosphorylated or acetylated p53 was detected with their corresponding antibodies.

Cell apoptosis. HEK 293 cells, H1299, and SNU475 cells were grown on glass coverslips in six-well tissue culture plate. When cells on the plate were 50% confluent, a transient transfection was done with 0.5 µg of green fluorescent protein–expressing vector pEGFPC3 (Clontech, Palo Alto, CA) together with a total of 3 µg of other plasmids including Myc-Daxx, Myc-Daxx{Delta}Axin, HA-Axin, HA-Axin{Delta}Daxx, Myc-p53, Myc-p53-R175H, pSUPER-Daxx, pSUPER-Axin, pSUPER-p53, and pSUPER-HIPK2 in different combinations. Cells were then stained with Hoechst 33342 and examined as previously described (28), and the remaining cells were scraped and lysed for Western blotting. For SNU-475 cells, Axin was introduced by using lentivirus infection. Briefly, 10 µg of pBOBI vector or pBOBI-Axin together with 10 µg of PMDL, 6 µg of VSV-G, and 4 µg of RSV-REV were transfected into 293T cells by using the calcium phosphate precipitation method. The lentiviral products were harvested thrice every 24 h, and were used to infect SNU-475 cells after concentration by centrifugation (29).

Immunofluorescent staining. HeLa cells were grown on glass coverslips in the cell culture medium described above for 16 h. Expression plasmids of Myc-Axin, HA-HIPK2, HA-Daxx, and Myc-Daxx were transfected into HeLa cells in different combinations as indicated where necessary. Approximately 24 h after transfection, cells were left untreated or irradiated with UV (80 J/m2), then cultured for another 6 h, and fixed with 3.7% formaldehyde-PBS for 10 min. The staining procedures were subsequently carried out as previously described (28), and visualized under a confocal laser scanning microscope (TCS SP2; Leica Microsystems, Inc., Bannockburn, IL).

Transcriptional reporter assay. p53-luc reporter (Stratagene, La Jolla, CA) was as described previously (28). PUMA-FRAG1-Luc and PUMA-FRAG2-Luc (30) were gifts from Dr. Vogelstein (The Johns Hopkins University, Baltimore, MD). HEK293 or H1299 cells were transfected in six-well dishes at 90% confluence with different reporters, 0.5 µg of LacZ expression plasmid and 0.5 µg of pEGFPN1, together with 2 µg of other plasmids including empty vector, Daxx, Daxx{Delta}Axin, Axin, Axin{Delta}Daxx, p53, pSUPER-Axin, pSUPER-p53, and pSUPER-HIPK2 in different combinations as indicated. All transfections were carried out in triplicate for at least five times, and error bars represent SD of the means.

Colony formation assays. HEK 293, SNU-475 (Axin–/–), U2OS, and SaOS-2 (p53–/–) cell lines were employed for colony formation assays. Cells were plated onto 60 mm dishes. When grown to 60% confluence, cells were transfected with empty pcDNA6 vector, pcDNA6-Daxx, or pcDNA6-Daxx{Delta}Axin individually. Approximately 48 h after transfection, drug-resistant cells were selected with fresh medium supplemented with 10 µg/mL of blasticidin for 3 weeks. Surviving colonies were fixed with 3.7% formaldehyde-PBS for 20 min at room temperature. After rinsing thrice with PBS, colonies were stained with 1% crystal violet in 20% ethanol.

Subcellular fractionation. Cell fractionation was done according to protocols as previously described (31, 32). Briefly, cells were collected and homogenized by 75 strokes in a 2 mL Kontes Douncer with the B-type pestle (Kontes Glass Company, Vineland, NJ) in an ice-cold homogenization buffer [250 mmol/L sucrose, 20 mmol/L Hepes-KOH (pH 7.4), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin]. Afterwards, cell lysates were centrifuged at 1,500 x g for 5 min at 4°C to remove the nuclei. The supernatant was then centrifuged at 17,000 x g for 15 min at 4°C and the resulting pellet was the mitochondrial fraction. Then, the supernatant was subjected to a second round of centrifugation at 16,000 x g for 20 min at 4°C and the remaining supernatant was the cytosolic fraction. The protein levels were measured by using the Bio-Rad Protein Array (Bio-Rad, Richmond, CA) and equal amounts of protein were analyzed by SDS-PAGE.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Daxx interacts with Axin in vivo and in vitro. To identify proteins that interact with Axin, we carried out yeast two-hybrid screen with a mouse fetal brain cDNA library using full-length Axin as bait in pGBKT7 vector (Clontech). Sequence analysis showed that one of the fish clones in pACT2 vector encoded the NH2-terminal 359 amino acid residues of Daxx as shown in Fig. 1A . To confirm the interaction between Axin and Daxx in mammalian cells, we cloned the full-length coding sequence of Daxx and did coimmunoprecipitation of Axin and Daxx in HEK 293T cells. HA-Daxx and Myc-Axin proteins were overexpressed in 293T cells, followed by reciprocal immunoprecipitation with anti-HA for Daxx and anti-Myc for Axin. The immunoblotting results showed that Daxx and Axin were coprecipitated with each other (Fig. 1B). We then tested whether endogenous Axin and Daxx could interact with each other using lysates of untransfected 293 cells with anti-Axin C2b (28) and anti-Daxx rabbit polyclonal antibody (for characterization of the antibody, see Supplementary Fig. S1). As shown in Fig. 1C, endogenous Axin and Daxx were also coimmunoprecipitated with each other, indicating that Axin forms a strong complex with Daxx in the cell.


Figure 1
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  		Figure 1. Daxx interacts with Axin in vivo and in vitro. A, yeast two-hybrid screening using full-length Axin as bait was done according to instructions from the manufacturer (Clontech). The "fish" clone contains a cDNA insert corresponding to amino acids 1 to 359 of Daxx as shown beneath the schema of full-length Daxx. B, 293T cells were cotransfected with HA-Daxx and Myc-Axin, and reciprocal coimmunoprecipitation was done with anti-HA (left) and anti-Myc (right). Daxx and Axin were detected in the corresponding immunoprecipitates by Western blotting with anti-HA and anti-Myc, respectively. C, immunoprecipitation of endogenous proteins from HEK293 cells was done separately with control IgG, rabbit anti-Axin C2b, and rabbit anti-Daxx polyclonal antibody, and immunoprecipitates along with total cell lysates (TCL) were analyzed separately by Western blotting with anti-Axin and anti-Daxx antibodies.

 
Next, we carried out mapping of their interaction interfaces, by generating a series of deletion mutants of Axin and Daxx as illustrated (Supplementary Fig. S2). Daxx was found to interact with full-length Axin, mutants D3, D4, and D6, but not with mutants D1, D2, D5, or D7, indicating that the region of amino acids 507 to 608 in Axin is critical for interaction with Daxx (Supplementary Fig. S2A). Daxx uses its NH2-terminal region (amino acids 1–197) for interaction with Axin, consistent with the presence of the NH2-terminal region of Daxx in the clone identified by a yeast two-hybrid screen (Supplementary Fig. S2B). These results show that there exist concrete domains in Axin and Daxx for their mutual interaction.

Axin tethers Daxx to p53 in a ternary complex. We previously showed that Axin possesses a domain for direct interaction with p53, in addition to association with p53 through HIPK2 (see ref. 28). In particular, one study showed that Daxx interacts with p53 (11), although another study contradicted that finding (10). We asked whether the complex of Axin and Daxx also contains p53. Myc-Axin and HA-Daxx were transfected into HEK293 cells, from which endogenous p53 was immunoprecipitated with the DO-1 anti-p53 antibody. When increasing amounts of Myc-Axin (0.5, 1.0, and 2.0 µg, respectively) were transfected, Daxx coimmunoprecipitated with p53 gradually increased (Fig. 2A ). These data suggested that Axin, Daxx, and p53 were copresent in the same complex. To formally establish that they actually form a ternary complex, we carried out a two-step coimmunoprecipitation (Fig. 2B). In this experiment, anti-HA was used to precipitate HA-Axin in the lysates of 293 cells that were cotransfected with Myc-Daxx and contain endogenous p53. Untagged Axin was transfected separately as a control. The precipitates were eluted with HA peptide. The eluates were then precipitated with the second antibody, anti-Myc (for Daxx), with IgG as a negative control. After the second round of immunoprecipitation, the components were analyzed by Western blotting using antibodies respectively for Axin, Daxx, and p53. From the total cell lysates expressing untagged Axin, no specific signal was detected in the final precipitate. HA-tagged Axin could coprecipitate both Daxx and p53, showing that Axin forms a ternary complex with Daxx and p53.


Figure 2
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  		Figure 2. Axin tethers Daxx to p53 in a ternary complex. A, Axin enhances the interaction between Daxx and p53 in a dose-dependent manner. HA-Daxx (1.0 µg) was cotransfected with increasing amounts of Myc-Axin (0.5, 1.0, and 2.0 µg, respectively) into HEK293 cells. Cell lysates were immunoprecipitated with anti-p53 (DO-1) for endogenous p53. Precipitates and total cell lysates were then immunoblotted with anti-Myc for Axin, anti-HA for Daxx, and DO-1 for p53, individually. B, two-step coimmunoprecipitation of the complex containing Axin, Daxx, and p53. Top, procedures of the two-step coimmunoprecipitation. Briefly, Myc-Daxx was transfected into 293 cells with HA-Axin or untagged Axin (as control). The first immunoprecipitation was done using anti-HA. The complex was eluted with HA peptide (Santa Cruz Biotechnology), followed by the second step of coimmunoprecipitation with anti-Myc for Daxx or control IgG. Immunoprecipitates from each step were blotted with anti-Myc, anti-Axin, and DO-1 for Daxx, Axin, and p53 protein levels, respectively. C, Axin mutants defective in binding to p53 or Daxx cannot enhance the interaction between p53 and Daxx. Blank vector, HA-Axin, HA-Axin{Delta}Daxx, and HA-Axin-M9 (lacking binding sites for p53 association, see ref. 28) were separately transfected with Myc-Daxx into 293 cells. Cell lysates were immunoprecipitated with DO-1 for endogenous p53, and samples were then analyzed by Western blot with anti-Myc, anti-HA, and DO-1 for protein levels of Daxx, Axin, and p53, respectively.

 
However, further characterization of the complex formation indicated that Daxx does not directly interact with p53, but through a bridge by Axin. As shown in Fig. 2C, Daxx was coprecipitated with p53 only in the presence of full-length Axin but not Axin{Delta}Daxx or Axin-M9 (lacking both sites for p53 association, see ref. 28). Similarly, in the presence of Axin, Daxx could coprecipitate only with wild-type p53 but not with the p53 mutant that is defective in Axin-binding (p53{Delta}Axin) (Supplementary Fig. S3A). In parallel, we found that p53 only associates with wild-type Daxx, but not with Daxx{Delta}Axin, which lacks an Axin-binding domain when cotransfected with Axin (Supplementary Fig. S3B). These results indicate that Daxx does not form a direct contact with p53, but associates with p53 through Axin, in accordance with the previous assertion that Daxx does not directly interact with wild-type p53 (10).

Daxx enhances p53 phosphorylation at Ser46 that requires Axin. We next examined whether Daxx also contributes to enhancement of p53 phosphorylation at Ser46 catalyzed by HIPK2 (33, 34). First, we found that Daxx indeed activated Ser46 phosphorylation of p53, but not Ser15 or Ser20, and that Axin and Daxx had an additive effect on p53 phosphorylation (Fig. 3A ). Daxx{Delta}Axin, which is defective in Axin binding, reduced approximately by half its ability to induce Ser46 phosphorylation compared with wild-type Daxx (Fig. 3B, left). Similarly, Axin{Delta}Daxx defective in association with Daxx exhibited reduced ability to stimulate p53 phosphorylation, indicating that maximal p53 phosphorylation requires both Axin and Daxx (Fig. 3B, right). We then tested whether Daxx-stimulated p53 phosphorylation at Ser46 was indeed mediated by HIPK2. The kinase-dead mutant HIPK2-K221R drastically attenuated Daxx-induced p53 phosphorylation (Fig. 3C). In addition, we generated a mutant HIPK2, HIPK2-{Delta}p53/{Delta}Axin, which lacks binding sites for both p53 and Axin but retains the binding site for Daxx. When coexpressed with Daxx, HIPK2-{Delta}p53/{Delta}Axin also abolished Daxx-induced phosphorylation of p53 (Fig. 3C). Consistently, the Axin mutant that is defective in binding to both of p53 and HIPK2 greatly retarded Daxx-induced p53 phosphorylation, whereas single removal of the binding sites of Axin for p53 and HIPK2 (Axin{Delta}p53 or Axin{Delta}HIPK2) gave rise to lesser reduction of the Daxx-induced p53 phosphorylation (Fig. 3D). The above results indicate that Daxx-induced p53 phosphorylation at Ser46 is mediated by HIPK2, and that the substrate p53 is bound by Axin and HIPK2. We also examined other posttranslation modifications of p53, such as acetylation of its COOH-terminal lysine residues. It was found that although p300 robustly enhanced p53 acetylation, Axin or Daxx was unable to induce acetylation of COOH-terminal lysine residues of p53, Lys320, Lys373, and Lys382 (Supplementary Fig. S4A).


Figure 3
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  		Figure 3. Daxx depends on Axin and HIPK2 to induce the phosphorylation of p53 at Ser46. A, Daxx and Axin additively stimulate the phosphorylation of p53 at Ser46, but not Ser15 or Ser20. H1299 cells were transfected with FLAG-p53, Myc-Daxx, and HA-Axin in different combinations as indicated. Phospho-p53-Ser46, Ser15, and Ser20 were detected with antibodies specific for each phosphorylated serine residue. Anti-Axin and anti-Daxx antibodies were used to probe Axin and Daxx, respectively. At short exposure (SE), transfected proteins were detected; longer exposures (LE) revealed low levels of endogenous Axin and Daxx. B, the Axin-binding domain of Daxx is critical for its activity to stimulate phosphorylation of p53 at Ser46 (left). FLAG-p53 was transfected into H1299 cells with Myc-Daxx or Myc-Daxx{Delta}Axin. Right, removal of Daxx-binding domain of Axin exhibited diminished ability to phosphorylate p53. H1299 cells were transfected with FLAG-p53 and HA-Axin or HA-Axin{Delta}Daxx. C, Daxx-induced phosphorylation of Ser46 on p53 was mediated by HIPK2. H1299 cells were transfected as indicated. At 30 h posttransfection, cells were harvested and the lysates were subjected to immunoprecipitation with anti-HA antibody for HA-p53 and followed by Western blotting. D, FLAG-p53, Myc-Daxx and different HA-Axin deletions were transfected into H1299 cells as indicated, followed by immunoprecipitation with anti-FLAG for p53 phosphorylation analysis as described above.

 
To further delineate the intricate complex formation and individual contribution of the Axin/Daxx/HIPK2 complex to p53 phosphorylation, we conducted pSUPER-based siRNA knockdown experiments. pSUPER-Axin (28) reduced Daxx-induced p53 phosphorylation (Supplementary Fig. S4B); pSUPER-Daxx diminished Axin-induced p53 phosphorylation (Supplementary Fig. S4C). pSUPER-HIPK2 (28) also attenuated Daxx-induced p53 phosphorylation (Supplementary Fig. S4D). These results again point to a requirement of HIPK2 for Daxx-mediated p53 phosphorylation and Axin contributes to the maximal activation of p53 by Daxx. Characterization of the efficiency and specificity of these pSUPERs was shown in Supplementary Fig. S5.

UV induces colocalization of Axin, Daxx, and HIPK2 in the nucleus. To visualize whether Axin, Daxx, and HIPK2 are subcellularly colocalized in the cell, we cotransfected Axin, Daxx, and HIPK2 alone or in combination into HeLa cells and carried out immunostaining. Axin is largely distributed in the cytoplasm, with Daxx and HIPK2 being exclusively present in the nucleus, regardless of single-transfection (data not shown) or cotransfection (Fig. 4 ). However, when the cells were exposed to UV irradiation, Axin was partially translocated into the nucleus and is overlapped with Daxx (Fig. 4A), and with HIPK2 (Fig. 4B). Daxx and HIPK2 are colocalized in the nucleus before or after UV treatment (Fig. 4C), in agreement with the previous report (14). Notably, when Axin was cotransfected with Daxx M1 mutant that is localized in the cytoplasm, Axin was also found colocalized with the mutant Daxx protein in the cytoplasm (Supplementary Fig. S6). Importantly, UV treatment seems to strengthen the interaction between Axin and Daxx, as determined by coimmunoprecipitation assay (Fig. 4D). All of these observations strongly indicate that Axin interacts with Daxx in the cell and form a ternary complex with HIPK2.


Figure 4
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  		Figure 4. The nuclear colocalization of Axin, Daxx, and HIPK2 could be induced by UV irradiation. A, UV treatment can induce Axin translocates from the cytoplasm into the nucleus and colocalizes with Daxx. HeLa cells were cotransfected with Myc-Axin and HA-Daxx. At 24 h posttransfection, cells were left untreated or irradiated with UV (80 J/m2). Eight hours after treatment, cells were fixed and analyzed by immunofluorescence staining using a mouse anti-Myc antibody and a rhodamine-conjugated rabbit anti-mouse secondary antibody for Axin (red), and a rabbit anti-HA antibody and a FITC-conjugated goat anti-rabbit secondary antibody for Daxx (green). Nucleus (blue) was displayed by staining of DNA with DAPI. Overlapping localization is shown in the merged images (yellow). B, UV treatment induces nuclear colocalization of Axin and HIPK2. HeLa cells were cotransfected with Myc-Axin (red) and HA-HIPK2 (green), followed by the same UV treatment and staining as described in (A). C, Daxx and HIPK2 are colocalized in the nucleus with or without UV irradiation. Cells were cotransfected with Myc-HIPK2 (red) and HA-Daxx (green), followed UV irradiation and staining. D, UV treatment can enhance interaction between Axin and Daxx. Myc-Axin and HA-Daxx were transfected into 293 cells. Approximately 24 h later, cells were left untreated or irradiated with UV (80 J/m2). After 8 h of additional culture, immunoprecipitation was done with anti-Myc for Axin, followed by Western blotting to detect Axin and Daxx.

 
Daxx cooperates with Axin to stimulate p53 transcriptional activity. Because Daxx has been shown to differentially modulate transcription from different p53-responsive promoters (11), we first tested whether Daxx could regulate the pathDetect p53-Luc reporter which carries the p53-specific enhancer element. When Daxx was cointroduced into 293 cells with the p53-Luc reporter, the luciferase activities were increased by ~6-fold, slightly less than the stimulation by Axin (Fig. 5A, left ). Cotransfection of Daxx and Axin further enhanced the transcriptional activity of the p53-Luc reporter (Fig. 5A, left), indicative of an additive effect by the two proteins. Daxx{Delta}Axin (mutant M5 as diagrammed in Supplementary Fig. S2) and Axin{Delta}Daxx (D7) exhibited a diminished ability to stimulate p53-dependent transcriptional activity (Supplementary Fig. S7A). Accordingly, siRNA against Axin, but not the control siRNA, attenuated the Daxx-mediated p53 transcriptional activity (Fig. 5A, right), showing that Daxx depends on Axin to activate p53 transcriptional activity. The siRNA against HIPK2, but not control siRNA, diminished the Daxx-induced transcriptional activity of p53 (Supplementary Fig. S7B), suggesting that HIPK2 is also required for the activation of p53 reporter stimulated by Daxx. Specific knockdown of p53 by pSUPER-p53 in 293 cells abolished the Daxx-induced activation of p53-dependent transcriptional activity (Supplementary Fig. S7C). Consistently, overexpressed Daxx failed to activate p53-Luc reporter in p53 null H1299 cells or H1299 cells overexpressing p53S46A (Supplementary Fig. S7E). However, in H1299 cells reintroduced with wild-type p53, Daxx exhibited a stimulatory effect on the reporter gene activity in a dose-dependent manner (Supplementary Fig. S7D), indicating that the Daxx-stimulated p53-Luc reporter activity was indeed mediated by p53.


Figure 5
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  		Figure 5. Daxx and Axin cooperatively stimulate p53 transcriptional activity. A and B, different p53-dependent luciferase reporters were transfected with other plasmids in various combinations as indicated into 293 cells. In each transfection, an equal amount of lacZ expression plasmid was introduced as internal control. Cells were harvested 30 h after transfection, followed by assay of luciferase activity. Columns, means of five independent experiments done in triplicate; bars, SD. A, Daxx and Axin can cooperatively stimulate p53-Luc reporter activity (left) and the siRNA against Axin diminished Daxx-induced p53 transactivation (right). 293 cells were transfected as indicated and cell lysates were measured for luciferase activity. B, both Daxx and Axin could enhance the transcriptional activity of PUMA-Luc and they have synergistic effects when coexpressed. C, Daxx and Axin induce cytochrome c release. 293 cells were transfected with HA-Axin or Myc-Daxx or both as indicated. At 30 h posttransfection, cells were collected and subjected to cell fractionation (M, mitochondria fraction; C, cytosol) as described in Materials and Methods. An equal amount of protein from each sample was analyzed by SDS-PAGE followed by Western blotting. Cells treated with valinomycin (at a final concentration of 10 µmol/L for 24 h) were included as a positive control. Hsp60 was detected as a loading control and a mitochondria marker for purity of subcellular fractions. Actin was also probed as cytosol loading control. D, neither Daxx nor Axin induced translocation of p53 into mitochondria. MCF-7 cells were transfected with Axin, Daxx, or both. Cells were treated with 5 µmol/L of camptothecin for 6 h or left untreated at 24 h posttransfection. Cells were then harvested and subjected to cell fractionation. Equal amounts of protein were analyzed by SDS-PAGE followed by immunoblotting separately with the indicated antibodies. Hsp60 validates equal mitochondrial loading. PML was used to exclude the contamination of the nuclear fraction.

 
To further explore the exact p53 target genes regulated by Daxx and Axin, a luciferase activity assay was done by using three additional reporter genes, PUMA, p21, and Bax reporters (28, 30). It was found that Daxx and Axin cooperatively stimulated the PUMA-FRAG1-Luc transcription activity (Fig. 5B), although they did not activate PUMA-FRAG2-Luc reporter that lacks the p53 binding sites in FRAG1-Luc (Supplementary Fig. S7F). However, as for p21 and Bax-Luc reporters, neither Daxx nor Axin could induce the luciferase activity (Supplementary Fig. S7G and H), suggesting that Daxx and Axin selectively enhance the transcriptional activity of p53 target genes.

Daxx and Axin induce cell apoptosis through cytochrome c release. It has been reported that the PUMA gene encodes two BH3 domain–containing proteins that are localized in the mitochondria (30, 35). In response to transactivation by p53, PUMA proteins are induced, which then form complex with Bcl-2 or Bcl-XL to induce cytochrome c release and cell apoptosis. Given that Axin and Daxx could stimulate PUMA gene transcription, we tested if they could induce cytochrome c release. The results showed that Daxx and Axin alone induced cytochrome c release when overexpressed in 293 cells (Fig. 5C). Moreover, when the two proteins were cotransfected, they showed a synergistic effect on cytochrome c release, indicating that Daxx and Axin cooperatively induce cell apoptosis through induction of PUMA, and subsequently, of cytochrome c release. On the other hand, transcription-independent induction of cell death by p53 has gained increasing attention (36, 37). In this way, p53 directly induce cytochrome c through translocation into the mitochondria, in which it forms inhibitory complexes with protective Bcl-2 and Bcl-XL. To address whether Axin and Daxx could also induce cell apoptosis through p53 transcription–independent pathways, we carried out experiments by isolating mitochondria from cells transfected with Axin, Daxx, or both, untreated or treated with camptothecin, and followed the detection of p53 by Western blot. Whereas camptothecin could effectively induce entry of p53 into the mitochondria, Axin or Daxx did not have such an effect. These results are shown in Fig. 5D, and indicate that Axin and Daxx most likely activate cell death through transcription-dependent pathways.

Inhibition of cell survival by Daxx requires endogenous Axin and p53. Daxx was shown to sensitize apoptosis induced by a variety of stimuli including UV (8), TGF-ß (9), arsenite trioxide, and IFN-{gamma} (38), and up-regulation of Daxx also mediates apoptosis in response to oxidative stress (39). We went on to assess any effect of Daxx on cell growth by performing clonogenic formation assay. For this assay, HEK293, SNU-475 (Axin–/–), U2OS, and SaOS-2 cells were used. In 293 and U2OS cells which contained functional p53, overexpression of Daxx{Delta}Axin that lacks the interaction domain for Axin did not inhibit cell growth compared with the wild-type Daxx that showed a strong inhibitory effect on colony formation, emphasizing that interaction of Daxx with Axin is important for Daxx-dependent inhibition of cell growth (Fig. 6A ). In SNU-475 cells lacking endogenous Axin and in p53-null SaOS-2 cells, Daxx failed to inhibit the clonogenic survival (Fig. 6A), consistent with the data from apoptosis assays which showed that both Axin and p53 are each crucial for Daxx-induced apoptosis (Supplementary Fig. S8). In the apoptosis assay, it was shown that specific knockdown of Axin, p53, or HIPK2 diminished Daxx-induced apoptosis in HEK293 cells (Supplementary Fig. S8A–C). Daxx displayed an attenuated ability to induce apoptosis in HEK293 cells expressing a dominant-negative form of p53 (R175H; Supplementary Fig. S8D), and failed to cause apoptosis in H1299 cells (Supplementary Fig. S8E), conforming to our conclusion that Daxx-induced apoptosis depends on p53. Conversely, in Axin-null SNU-475 cells, p53 induction of apoptosis was severely compromised unless Axin was reintroduced by lentivirus infection, whereas siRNA against Daxx reduced p53-dependent cell death even in cells with reintroduced Axin (Fig. 6B, left). Similarly, p53 also needs both endogenous Axin and Daxx to gain maximal ability to induce cell death in H1299 cells as depletion of endogenous Daxx or Axin reduced p53-induced cell apoptosis (Fig. 6B, right). Moreover, when Axin or Daxx were knocked down by its specific siRNA, fewer cells were found to undergo apoptosis after UV treatment, and when both Axin and Daxx were knocked down, UV-induced cell death was further decreased (Fig. 6C).


Figure 6
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  		Figure 6. Daxx and Axin cooperate to induce cell apoptosis. A, Axin-binding domain of Daxx is critical for it to inhibit cell growth. HEK 293, SNU-475 (Axin–/–), U2OS, and SaOS-2 (p53–/–) cell lines were used to perform colony formation assay. Each cell line was transfected with empty pcDNA6 vector, Daxx, or Daxx{Delta}Axin. At 48 h posttransfection, cells were selected with medium supplemented with 10 µg/mL of blasticidin for 3 wks and the surviving colonies were fixed and stained with crystal violet. B, p53 depends on reintroduced Axin and Daxx to induce a maximal rate of cell death in SNU-475 cells (left). Axin was reintroduced by a lentivirus system (pBOBI); empty virus–infected cells (blank columns, control) and pBOBI-Axin-infected cells (black columns). Columns, means of five independent experiments; bars, SD. Statistical analyses were done using t test. *, P < 0.01 compared with control cells infected with empty virus (blank columns); #, P > 0.05 compared with control cells (blank columns); **, P < 0.01 compared with the cells in which p53 and Axin were simultaneously introduced (second and fourth black columns). Right, Daxx and Axin cooperatively enhance p53-induced cell apoptosis in H1299 cells. Inset, Western blots indicate expression levels of each transfected construct, with green fluorescent protein (GFP) as internal control for transfection efficiency. Columns, means of five independent experiments; bars, SD; *, P < 0.01 compared with control cells transfected with empty pCMV5 vector (blank columns); **, P < 0.01 compared with the cells transfected with p53 alone (first black column) or transfected with p53 and RNAi control (second black column); +, P < 0.01 compared with cells transfected with p53 and pSUPER-Daxx (third black column) or cells transfected with p53 and pSUPER-Axin (fourth black column). C, knockdown of Daxx and Axin by siRNA could attenuate UV-induced cell apoptosis in 293 cells. Cells were transfected as indicated, and at 24 h posttransfection, cells were treated with UV at 80 J/m2 (black columns). After another 8 h of culture, cells were subjected to apoptosis assay as described previously (28). Columns, means of five independent experiments; bars, SD; *, P < 0.01 compared with control cells not treated with UV (blank columns); **, P < 0.01 compared with the cells treated with UV alone (first black column) or the cells transfected with RNAi control, followed by UV irradiation (second black column); +, P < 0.01 compared with cells transfected with pSUPER-Daxx and followed by UV treatment (third black column), or cells transfected with pSUPER-Axin, followed by UV irradiation (fourth black column). D, schematic representation of possible modes of HIPK2-mediated activation of p53 by Axin and Daxx. As discussed in the text, Daxx can activate p53 with HIPK2 either alone or in conjunction with Axin/HIPK2/p53 complex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have provided strong evidence for a physical and functional interaction of Axin, Daxx, HIPK2, and p53. We show that Daxx does not form direct contact with p53, but that Axin serves as a bridge to tether Daxx to p53. Importantly, the association between Axin and Daxx is enhanced in cells after UV treatment, which indicates that the complex formation is physiologically regulated. Whereas Daxx alone can activate p53 that is presumably bound with HIPK2, Axin significantly augments such an effect, suggesting that Axin/Daxx/HIPK2/p53 complex formation leads to maximal stimulation of p53 activities, at least with respect to transcriptional activation and cell growth inhibition. The physiologic relevance of our novel finding can be further manifested by the observation that Axin and Daxx interact with each other at their endogenous levels. Moreover, Daxx-mediated inhibition of cell growth depends on the presence of both functional Axin and p53, as Daxx fails to induce cell death in Axin–/– cells or p53-deficient SaOS-2 cells in clonogenic assays. Knockdown of Axin or Daxx severely compromise UV- or p53-induced apoptosis, consistent with the notion that Axin/Daxx complex formation is needed to induce maximal activation of p53.

It was previously shown that Daxx interacts with HIPK2, and upon TGF-ß treatment, HIPK2 phosphorylates Daxx which, in turn, leads to JNK activation (15). Our results clearly established that Axin, Daxx, and p53 form a ternary complex that promotes HIPK2 phosphorylation of p53 at Ser46. Knockdown of Axin by siRNA significantly reduced Daxx-induced p53 phosphorylation; Daxx{Delta}Axin defective in Axin-binding displays a much compromised ability to induce p53 phosphorylation. Similarly, when Daxx was knocked down or when its Daxx-binding domain was deleted, Axin exhibited reduced activity towards activation of phosphorylation or enhancement of transcriptional activity of p53. Based on all these observations, it is legitimate to suggest that Axin and Daxx seem to adopt both parallel routes and a convergent means to activate p53 (Fig. 6D). In either case, HIPK2 seems to be the protein kinase that catalyzes the Ser46 phosphorylation. Daxx alone can interact with, and activate, HIPK2 leading to increased phosphorylation of p53. Under certain physiological conditions or in the presence of stress stimuli such as UV, Axin is translocated into the nucleus to form Axin/Daxx/HIPK2/p53 complex that yields a higher stimulation of p53 than Daxx/HIPK2/p53 or Axin/HIPK2/p53. It is therefore conceivable that cellular context with regard to Axin abundance in different cell lines can be an important factor when assaying for the ability of Daxx to induce cell death.

Our current work has also established that Axin and Daxx stimulates the transcriptional activation of proapoptotic p53 target genes. Interestingly, Axin and Daxx display strong selectivity in boosting p53-dependent genes. Among the reporter genes tested, including PUMA, p21, and Bax, only the PUMA reporter gene is activated. We also found that Axin and Daxx could induce cytochrome c release, in accordance with the induction of the PUMA gene by the two proteins. However, we did not see a direct translocation of p53 into the mitochondria to cause the release of cytochrome c, in contrast to several reports showing that gamma irradiation can induce translocation of p53 into mitochondria to permeabilize the outer membrane. Rather, Axin/Daxx-induced apoptosis seems to adopt a transcription-dependent route, by activating proapoptotic genes such as PUMA that are mitochondrial proteins and inhibit antiapoptotic Bcl-2 or Bcl-XL. Complex formation of PUMA with Bcl-2, in turn, causes the release of cytochrome c to initiate the activation of the apoptotic cascade which involves Apaf-1 (37). In sum, our results have provided a mechanistic insight into how Daxx cooperates with other cellular factors to stimulate the multifaceted function of p53 as a tumor suppressor.


   Acknowledgments
 
Grant support: National Natural Science Foundation of China (nos. 90208015, 30500273, 30528014, and 30370306) and grants from Hong Kong Research Grant Council (HKUST6127/04M, HKUST6416/05M). This work was also supported by "Project 111" sponsored by the State Bureau of Foreign Experts and Ministry of Education (no. B06016). S-C. Lin is a recipient of a National Outstanding Young Investigator Award (no. 30125012).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. B. Vogelstein (The Johns Hopkins University) for providing PUMA luciferase reporters.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/ 8/06. Revised 10/ 4/06. Accepted 10/19/06.


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