WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53*

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

The human WWOX gene, which comprises nine exons encoding the WWOX/WOX1 family proteins, is located on a fragile site on the chromosome ch16q23.3-24.1 (6 -9). Eight mRNA transcripts of the WWOX gene have been found so far (7). However, it is still not known whether all of the mRNA transcripts are translated successfully into proteins. Isoforms WWOXv1 (46 kDa), WWOXv2 (42 kDa), and WWOXv8 (60 kDa) and several other small proteins can be found in normal and several types of cancer cells (3, 8 -11). 3 Genetic alterations of the WWOX gene and disappearance of WWOX/WOX1 family proteins have been shown in multiple types of cancers, especially at an invasive or a metastatic stage (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Hypermethylation of the WWOX gene may inactivate its expression (21). In contrast, significant up-regulation of WWOX/WOX1 family proteins has been shown during progression of breast, prostate and other cancers to a premetastatic state (10,22,23). Also, absent expression of these family proteins in metastatic cancer cells is not necessarily due to disruption of the WWOX gene. We have recently determined that post-transcriptional blockade of the fulllength mRNA translation to protein may account for the disappearance of the WWOX/WOX1 family proteins in cutaneous squamous cell carcinoma cells in patients and in UVB-treated mice (24). Wild type WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence and a C-terminal short-chain alcohol dehydrogenase/reductase domain (which contains a mitochondria-targeting sequence) (3). Sex steroid hormones such as estrogen and androgen activate WOX1 via Tyr 33 phosphorylation (p-WOX1) and nuclear translocation (11). Importantly, p-WOX1 is located in the mitochondria during benign prostatic hypertrophy (11). Nuclear translocation of p-WOX1 occurs when prostate cells progress toward cancerous and metastatic states (11), suggesting a critical role of WOX1 phosphorylation during prostate cancer development.
We determined that WOX1 enhances the cytotoxic function of tumor necrosis factor (TNF) 4 and induces apoptosis when overexpressed (3). We also showed that in response to stress or apoptotic stimuli, WOX1 becomes phosphorylated at Tyr 33 , which allows its complex formation with activated p53 and JNK1 (25). The p53-WOX1 complex translocates to the mitochondria and nuclei to mediate apoptosis (25,26). WOX1 induces apoptosis synergistically with p53 (3,25). In contrast, JNK1 may block WOX1-induced cell death (25). Src is known to phosphorylate WOX1 at Tyr 33 (27). We also determined that Tyr 33phosphorylated WOX1 translocates to the mitochondria and nuclei during light-induced degeneration of photoreceptors in rat eyes, as determined by light and electron microscopy (28).
In this study, we further characterized the binding interaction between p53 and WOX1. We determined that WOX1 plays an essential role in TNF-, UV light-, staurosporine-, and p53-mediated cell death and that Tyr 33 -phosphorylated WOX1 binds and regulates the stability of Ser 46 -phosphorylated or activated p53.
Antibodies and Antibody Production-We have generated specific antibodies against a Tyr 33 -phosphorylated peptide of WOX1 (25) and a recombinant full-length WOX1 protein, tagged with glutathione S-transferase (11). The quality of these antibodies has been documented (10,11,24,25,28). In addition, we constructed the N-terminal WW domain regions of murine WOX1 in pGEX-2T (Amersham Biosciences) and expressed the fusion protein tagged with glutathione S-transferase in bacteria. Antibody against the purified glutathione S-transferase-WOX1ww was performed in rabbits as described (3,10,11). This antibody recognized a 46-kDa WOX1 as determined in Western blotting using L929 and other cells and was shown to be effective for co-immunoprecipitation (see "Results").
Where indicated, L929 and other cell lines were electroporated with the above constructs (200 V, 50 ms; Square Wave BTX ECM830; Genetronics), cultured overnight, and then treated with bovine testicular hyaluronidase PH-20 (Sigma) for the indicated times. Alternatively, the cells were exposed to UV irradiation (240 mJ/cm 2 ; using a UV crosslinker from Fisher), followed by culturing for 1 h. The cells were then subjected to extraction and separation of cytosolic and nuclear fractions using an NE-PER nuclear and cytoplasmic extraction reagent (Pierce). The extent of protein expression was determined using the indicated specific antibodies.
In addition, we generated stable transfectants using the above transfected cells by exposure to G418 (up to 500 g/ml) for 2 weeks (3,10,25). The extent of protein expression was examined by fluorescence microscopy and Western blotting.
siRNA Targeting WOX1 and Stable WOX1 Knockdown L929 Cells-We have previously constructed a siRNA-expressing plasmid (in pSup-pressorNeo vector; Imgenex), targeting the expression of human and mouse WOX1 (10). A scrambled sequence construct in the pSuppres-sorNeo plasmid was used as a control (10). Stable transfectants of L929 cells were also established using G418 selection (10). The extent of WOX1 protein knockdown was examined by Western blotting using our specific antibodies. We have also established stable WOX1 knockdown of neuroblastoma SK-N-SH cells (10). These cells were also used for experiments.
Where indicated, we used retroviral expression of WOX1si in human and murine cell lines to knock down the expression of WOX1 (10). In controls, an empty retrovirus was used (10).
Co-immunoprecipitation-Co-immunoprecipitation was performed as previously described (3,10,11,25,29). Briefly, Molt-4 T cells or other indicated cell lines were grown overnight (in 150-mm Petri dishes) and exposed to UV light (or other indicated reagents), followed by culturing for 1 h. Both cytosolic and nuclear fractions were prepared using the NE-PER nuclear and cytoplasmic extraction reagent (Pierce) and quantified (Bio-Rad protein assay). 3-4 g of anti-p53 or WOX1 IgG were added to these protein preparations (ϳ0.5-0.7 g in 500 ml of NE-PER extraction buffer), followed by rotating end-over-end at 4°C for 5-16 h. The mixtures were then added to 20 l of protein A-agarose beads (Pierce), rotated for 2 h at 4°C, washed with Tris-buffered saline containing 0.5% Nonidet P-40, and processed for SDS-PAGE (3). The presence of WOX1, p53 and other indicated proteins in the precipitates was determined by Western blotting, using specific antibodies. Where indicated, nonimmune serum or IgG was used in negative controls.
p53 Stability-p53-deficient H1299 cells, suspended in Dulbecco's modified Eagle's medium containing 0.5% albumin, were electroporated with DNA constructs of p53-pDsRedN1 or p53⌬46-pDsRedN1 in the presence or absence of a scrambled pSuppressorNeo or WOX1si-pSup-pressorNeo (25 g DNA each; 200 V, 50 ms; Square Wave BTX ECM830). We found that albumin could significantly increase the transfection efficiency by at least 300 -500%. The cells were cultured 48 h and treated with cycloheximide (CHX; 300 M) for 0, 20, 40, and 60 min. The cells were then harvested, lysed, and subjected to SDS-PAGE and Western blotting for determining the stability of p53-DsRed and p53⌬46-DsRed. In some experiments, cells were treated with CHX in the presence or absence of a proteasome inhibitor zLLL or MG132 (Biomol), or the indicated cells were pretreated with or without nutlin-3 (10 M) overnight to inhibit MDM2, followed by examining the binding of p53 with WOX1 and/or MDM2.

RESULTS
Binding of Ser 46 -phosphorylated p53 with Tyr 33 -phosphorylated WOX1-We determined the characteristics of p53 and WOX1 binding interactions. Exposure of Molt-4 T cells to UV light resulted in a timedependent phosphorylation of p53 at Ser 46 and WOX1 at Tyr 33 and their nuclear translocation (Fig. 1A). Ser 46 phosphorylation plays a critical role in p53-induced apoptosis and gene transcription (30 -32). Thompson et al. (33) showed that under the inhibition of MDM2 by nutlin-3, phosphorylation of p53 on key serines is not essential for transcriptional activation and apoptosis. UV light also increased the levels of nuclear histone H3 and its acetylation at lysine 9 (H3k9) (Fig. 1A). Immunoprecipitation using anti-p53 antibody showed that there was an increased binding of p53 with WOX1 in UV-irradiated Molt-4 T cells (Fig. 1A). Precipitating with anti-WOX1 IgG also showed an increased binding of WOX1 with p53 (data not shown; or see Fig. 8 from using HCT116 cells). Phosphorylation of p53 at other sites such as Ser 15 was also observed (data not shown). UV light-induced nuclear translocation of p53 and WOX1 was further confirmed by immunofluorescence microscopy (Fig. 1B). Similarly, exposure of L929 fibroblasts to UV light resulted in Tyr 33 phosphorylation and nuclear translocation of p53 and WOX1, along with p53. (see supplemental Fig. 1).
Under similar conditions, UV light increased phosphorylation of p53 at Ser 46 and WOX1 at Tyr 33 in monocytic U937 cells (Fig. 1C). An increased binding of p53 with p-WOX1 was observed in UV-irradiated U937 cells (Fig. 1C).
IB␣ binds a portion of cytosolic p53, and this interaction appears to stabilize p53 from degradation (29,34). Exposure of neuroblastoma SK-N-SH cells to genotoxic stress, such as UV light, anisomycin, and etoposide, also caused phosphorylation and nuclear translocation of p53 and WOX1 and their binding interaction (data not shown). IB␣ was also present in the cytosolic p53-WOX1 complex.
Similarly, hypoxic stress increased the binding of p53 with WOX1. SK-N-SH cells were exposed to deferroxamine for 1 h to induce hypoxic stress (29) and were shown to have an increased binding of WOX1 with p53, as determined by immunoprecipitation with anti-WOX1 IgG (Fig. 1D).
We mapped the domain regions and critical amino acid residues responsible for binding of p53 with WOX1, using a CytoTrap yeast two-hybrid analysis for protein/protein interaction in the cytoplasm (3,10,25,29). In agreement with our previous observations (3), human p53 bound to the N-terminal WW domains of murine WOX1, as evidenced by the presence of surviving yeast colonies growing at 37°C on selective galactose plates ( Fig. 2A). We have determined that WOX1 interacts with an ectopically expressed N-terminal proline-rich segment in p53 (amino acids 66 -100) (3). However, deletion of this segment from the full-length p53 could not completely abrogate binding of p53 with WOX1 ( Fig. 2A), suggesting that other critical regions or amino acid residues are involved in the binding. Interestingly, deletion of the conserved phosphorylation site Ser 46 in p53 significantly reduced its binding with the full-length WOX1 or the N-terminal WW domains of WOX1 ( Fig. 2A). In contrast, deletion of other critical phosphorylation residues such as Thr 18 and Ser 20 in p53 could not abolish its binding with WOX1 ( Fig. 2A). In positive controls, self-binding of MafB is shown ( Fig. 2A). In negative controls, no binding interactions were observed for empty vectors and collagenase versus lamin C ( Fig. 2A). A, Molt-4 T cells were exposed to UV light (240 mJ/cm 2 ), followed by culturing for 10 -120 min. Both cytosolic and nuclear fractions were prepared and examined for phosphorylation of p53 at Ser 46 and WOX1 at Tyr 33 (p-WOX1) and their nuclear translocation. UV light also increased the levels of nuclear histone H3 and its acetylation at lysine 9 (H3k9). By immunoprecipitation (IP) using p53 IgG antibody, UV light was shown to induce binding of cytosolic p53 with WOX1 in Molt-4 T cells. B, we confirmed the UV light-induced nuclear translocation of p53 and WOX1 in Molt-4 cells by immunofluorescence microscopy. Cells were stained with IgG antibodies against WWOX (N19; goat) and full-length p53 (FL-393; rabbit), respectively (3,4). Secondary staining was performed using anti-goat IgG (conjugated with Texas Red) and anti-rabbit IgG (conjugated with Cy2). Nuclei were stained with 4Ј,6-diamidino-2-phenylindole. C, monocytic U937 cells were exposed to UV light (240 mJ/cm 2 ) and shown to have increased phosphorylation of cytosolic p53 at Ser 46 and WOX1 at Tyr 33 , and p53 binding with p-WOX1. D, similarly, neuroblastoma SK-N-SH cells were exposed to deferroxamine (500 M) for 1 h to induce hypoxic stress and shown to have an increased binding of p53 with WOX1.
We examined WOX1 phosphorylation at Tyr 33 in the above mentioned yeast cells. These cells were grown at room temperature for 2 days in a galactose-containing broth (to reach an exponential phase). To induce the activation of Ras pathway, some cells were then cultured at 37°C for 1 h. Yeast cell proteins were extracted using the Y-PER lysis buffer. By Western blotting, we showed that heat induced the expression of p53 and WOX1 and its phosphorylation at Tyr 33 (p-WOX1) in yeast cells transfected with wild type p53 and WOX1 constructs (Fig.  2B). In contrast, no WOX1 phosphorylation was observed in cells transfected with p53⌬S46 mutant and wild type WOX1 (Fig. 2B). Together, these results suggest that WOX1 phosphorylation occurs during its binding with p53.
To further confirm the above observations, p53-deficient H1299 cells were transfected with wild type p53 or p53⌬S46, tagged with DsRed. By co-immunoprecipitation using antibodies against p-WOX1 (25), we determined that UV light induced physical association of p-WOX1 with the wild type p53-DsRed but not p53⌬S46-DsRed (Fig. 3). These results were reproducible using L929 and SK-N-SH cells (data not shown). These observations clearly indicate that Ser 46 phosphorylation in p53 is essential for binding with Tyr 33 -phosphorylated WOX1.
WOX1 Is Essential for TNF-, UV Light-, Staurosporine-, and p53mediated Cell Death-We have recently generated plasmid and retroviral vectors for expressing small interfering RNA (siRNA)-targeting WOX1 (WOX1si) (10). Murine L929 cells were transfected with a WOX1si or a scrambled siRNA plasmid construct by electroporation. Stable transfectants were selected using G418. WOX1si-expressing cells had a significantly reduced expression of WOX1 protein (Ͼ50%), compared with control cells (Fig. 4A). We have determined that WOX1 enhances TNF cytotoxicity in L929 cells (3). Expectedly, suppression of WOX1 expression by siRNA resulted in cellular resistance to TNF (Fig.  4A). Similarly, these WOX1si-expressing cells resisted staurosporineand UV light-induced cell death (Fig. 4, B and C). Similar results were observed when challenging WOX1si-expressing SK-N-SH cells to UV light and staurosporine (data not shown). In parallel, we have recently determined that suppression of WOX1 by siRNA in cutaneous squa-  Mapping of p53 and WOX1 interactions by yeast two-hybrid analysis and temperature-dependent WOX1 phosphorylation. A, analysis of protein/protein interaction in the cytoplasm was performed using a Ras rescue-based yeast two-hybrid system (3,10,25,29). In positive controls, binding of WOX1 with p53 and MafB selfinteraction are shown, as evidenced by the growth of yeast at 37°C using selective agarose plates containing galactose. In negative controls, no yeast growth at 37°C was observed for the empty pSos/pMyr vectors and collagenase and lamin C. Human p53 physically interacted with the N-terminal WW domains of murine WOX1. Deletion of an N-terminal proline-rich segment (amino acids 66 -100; see block No. 2) from the full-length p53 did not abrogate the binding. Similarly, deletion of Thr 18 or Ser 20 in p53 could not prevent its binding with WOX1. In contrast, deletion of Ser 46 in p53 significantly reduced the binding. Alterations of Tyr 33 to Arg in the first WW domain of WOX1 abolished its binding with p53. p53 structure was as follows: transcriptional activation domain (1); proline-rich region (2); DNA-binding domain (3); tetramerization domain (4). B, to examine WOX1 phosphorylation at Tyr 33 , some yeast cells were grown at room temperature for 2 days in a galactose-containing broth (to reach a preplateau stage), followed by culturing at 37°C for 1 h to induce the activation of Ras pathway. Heat induced the expression of p53, WOX1, and its phosphorylation at Tyr 33 (p-WOX1) in yeast cells transfected with wild type p53 and WOX1. In contrast, no WOX1 phosphorylation was observed in cells transfected with p53⌬S46 mutant and wild type WOX1. DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 mous cell carcinoma SCC-25 and keratinocyte HaCaT cells enables these cells to resist UVB-mediated apoptosis (24).

WOX1 and p53 Interactions
We have shown that functional suppression of WOX1 by antisense mRNA or dominant negative WOX1 abolishes p53-mediated cell death (3,25). Similarly, p53-deficient H1299 cells were electroporated with p53-DsRed or DsRed vector only and simultaneously cultured in the presence of retrovirus (expressing siRNA-targeting WOX1) for 24 h. The retroviral expression of WOX1si was prepared as described (10). Ectopic p53-DsRed-mediated death of H1299 cells was abolished in the presence of WOX1si (Fig. 5). In empty retrovirus-transfected control cells, p53 effectively induced cell death (Fig. 5). The extent of WOX1 knockdown was ϳ60%. Again, these results are in agreement with our previous observations (3,25).
UV light activates p53 and WOX1 in various cultured cell lines (25,29,38). Similarly, UV light rapidly induced p53 expression in the control cells but not in the WOX1si-expressing cells (Fig. 6B). In addition to UV light and PH-20, we also determined that apoptosis-inducing chemicals, such as anisomycin and etoposide, could not induce p53 expression in these WOX1 knockdown cells (data not shown).
We have previously designed a dominant negative WOX1 (dn-WOX1; tagged with EGFP), which blocks stress stimuli-induced WOX1 phosphorylation at Tyr 33 as well as p53-mediated apoptosis (25). Failure of induction of p53 protein expression in WOX1 knockdown cells is probably not due to interference of p53 mRNA by WOX1si. We established stable transfectants expressing dn-WOX1 (and EGFP as controls). Again, these dn-WOX1-expressing cells resisted UV-induced p53 expression, compared with control cells (Fig. 6C; data not shown for the PH-20 induction). Similarly, COS7 cells express a high level of

. Suppression of WOX1 expression by siRNA induces resistance to death by TNF, staurosporine, and UV light in L929 cells.
A, we established stable L929 cell lines expressing scrambled siRNA or WOX1 siRNA (WOX1si) (constructed in pSuppressorNeo) (10). Compared with the "scrambled" control cells, the WOX1si-expressing cells resisted TNF-mediated cell death during exposure for 16 -24 h (n ϭ 8; mean Ϯ S.D.; p Ͻ 0.001, Student's t tests). Inhibition of WOX1 protein expression was more than 50% in the WOX1si-expressing cells. The extent of cell death was determined by crystal violet staining. Similar results were observed using non-radioactive cell proliferation assay (Promega; data not shown). B, these WOX1si-expressing cells also resisted staurosporineinduced cell death during exposure to staurosporine for 16 h (n ϭ 8; mean Ϯ S.D.; p Ͻ 0.01, Student's t tests). C, compared with "scrambled" control cells, WOX1si-expressing cells resisted UV light-induced DNA fragmentation or apoptosis. Both established cells were exposed to UV light (720 mJ/cm 2 ), followed by culturing for 16 h and then a processing DNA fragmentation assay (3).  (2)). Ectopic p53-DsRedmediated death of H1299 cells was inhibited in the presence of WOX1si (n ϭ 8; mean Ϯ S.D.). Similar results were observed from a 48-h culture (data not shown). FIGURE 6. Absence of p53 expression in hyaluronidase PH-20-or UV-treated WOX1 knockdown cells. A, stable L929 cells, expressing a WOX1si or a scrambled siRNA, were established. WOX1 protein level was reduced by ϳ50% in these cells. Compared with scrambled control cells, no induction of p53 expression in WOX1si-expressing cells was observed when these cells were exposed to PH-20 (200 units/ml) for 30 min. PH-20 induces p53 expression in L929 cells (3,4,37). B, these cells were exposed to UV light, followed by culturing for 30 min at 37°C and examination of p53 expression. Compared with control cells, UV light could not restore p53 and WOX1 expression in the WOX1siexpressing cells (data not shown for WOX1 expression). C, stable L929 transfectants, expressing dn-WOX1 (tagged with EGFP) or a control EGFP vector, were established. UV light could not induce p53 protein expression in the dn-WOX1-expressing cells (data not shown for the PH-20 induction). D, the reduced p53 expression was not due to downregulation of p53 mRNA in WOX1 knockdown cells, as determined by RT-PCR. endogenous p53. dn-WOX1 suppressed p53 expression by 70 -80%, and UV light could not restore p53 expression (data not shown).
By RT-PCR, we showed that failure of UV light-induced p53 protein expression was not due to down-regulation of p53 mRNA in the WOX1si-expressing cells (Fig. 6D). Also, UV light induced WOX1 gene expression in control cells but not in the WOX1si-expressing cells (Fig.  6D). Similar results were observed by testing SK-N-SH cells expressing scrambled or WOX1 siRNA (data not shown).
p53 Is Destabilized in WOX1 Knockdown Cells-p53-deficient lung H1299 cells were electroporated with p53-DsRed in the presence of a scrambled or WOX1 siRNA construct (10). p53-DsRed is metabolically degradable. These cells were grown for 48 h and treated with CHX to block protein synthesis for indicated times. Cells were harvested and analyzed for the stability of p53-DsRed. The results showed that p53-DsRed was relatively stable in control cells expressing scrambled siRNA (Fig. 7A). In contrast, the stability of p53-DsRed was significantly reduced in the WOX1si-expressing cells (Fig. 7A). Approximately, 70% reduction of WOX1 was shown in the WOX1si-expressing cells. In additional experiments, we showed that ectopic p53⌬S46-DsRed was relatively stable in the presence or absence of WOX1 (Fig. 7B). WOX1 could not bind p53⌬S46-DsRed (Fig. 3).
The enhanced degradation of p53 was due, in part, to increased ubiquitination in the absence of WOX1. Breast MDA-MB-231 cells were cultured in the presence of empty or WOX1si retrovirus for 48 h, followed by treating with zLLL (MG132) for 2 h. Suppression of WOX1 expression by siRNA induced spontaneous ubiquitination of p53, and zLLL further stimulated accumulation of ubiquitinated p53 in these cells (Fig. 7C). MDA-MB-231 cells expressed mutant p53 (39). The reason for using these cells is that in the absence of WOX1, mutant p53 was relatively stable (Fig. 7C), which allows analysis of p53 ubiquitination. In contrast, wild type p53 was not stable in the absence of WOX1 (Fig. 6), which made it difficult for assaying ubiquitination of wild type p53.
We also show the presence of p53-MDM2-WOX1 complex in COS7 fibroblasts, as determined by co-immunoprecipitation using anti-WOX1 IgG (see supplemental Fig. 2). Several MDM2 isoforms were present in COS7 cells. However, only a 42-kDa MDM2 interacted with p53-WOX1 (see supplemental Fig. 2). Under similar conditions, UV light induced complex formation of p53, MDM2, and WOX1 in Molt-4 FIGURE 7. Reduced p53 stability in the absence of WOX1. p53-deficient lung H1299 cells were electroporated with p53-DsRed or p53⌬S46-DsRed, in the presence of a scrambled or WOX1 siRNA (WOX1si) construct. After culturing for 48 h, these cells were treated with CHX (300 M) to block protein synthesis for the indicated times. A, p53-DsRed was relatively stable in control cells expressing scrambled siRNA (less than 15% reduction). In contrast, the stability of p53-DsRed was significantly reduced in the WOX1si-expressing cells (ϳ30 -40% reduction). B, the stability of p53⌬S46-DsRed was relatively unchanged in the presence or absence of WOX1si. C, breast MDA-MB-231 cells were exposed to empty or WOX1si retrovirus for 48 h and then treated with zLLL (MG132) for 1 h. Suppression of WOX1 expression by siRNA induced spontaneous ubiquitination of p53, and zLLL further stimulated the accumulation of ubiquitinated p53 in these cells. In the right panel, an average of two experiments is shown.
T cells and COS7 and L929 fibroblasts (data not shown). Also, nutlin-3 dissociated MDM2 from the complex of p53 and WOX1, and p53 resisted CHX-mediated degradation in these cells (data not shown).
Together, the above data show that UV induces activation of p53 and WOX1 and their complex formation in the presence of MDM2. In contrast, nutlin-3-activated p53 binds WOX1, but not MDM2, suggesting that there are two types of p53 activation forms, one resistant to MDM2 binding and the other one susceptible.
In summary, UV light induces phosphorylation of p53 at Ser 46 and WOX1 at Tyr 33 and their complex formation in the presence of MDM2 (Fig. 10A). Inhibition of MDM2 by nutlin-3 stabilizes p53 and its interaction with WOX1. This p53 appears to be conformationally altered. UV light restores the complex formation of p-p53-p-WOX1-MDM2 in nutlin-3-treated cells. WOX1 alone binds MDM2 probably via its C-ter-minal short-chain alcohol dehydrogenase/reductase domain. Also, nutlin-3 dissociates the binding, whereas UV light restores the binding (Fig.  10B).

DISCUSSION
In this study, we have demonstrated for the first time that WOX1 is involved in binding and stabilizing p53. During stress response, both p53 and WOX1 undergo phosphorylation at Ser 46 and Tyr 33 , respectively, and the phosphorylation is essential for their binding. Ser 46 phosphorylation is involved in p53-mediated apoptosis and gene transcription (30 -32). WOX1 and p53 induce apoptosis in a synergistic manner (3). p53-induced apoptosis is blocked when WOX1 phosphorylation or protein expression is blocked by dn-WOX1 or antisense mRNA (3,25). We further demonstrate here that knockdown of WOX1 protein expression by siRNA enables L929 cells to resist to apoptosis by TNF, FIGURE 8. WOX1 physically interacts with p53 and MDM2 and nutlin-3 dissociates MDM2 from the complex. A, by co-immunoprecipitation, WOX1 was shown to physically interact with both p53 and MDM2 (90, 60, and 42 kDa) in colorectal HCT116 cells. Nutlin-3 (10 M) induced release of MDM2 from the p53-WOX1 complex in these cells during treatment for 16 h. B, in contrast, UV light enhanced the binding of p-WOX1 with p53 and MDM2 in HCT116 cells (i.e. HCT116 cells were exposed to UV light (240 mJ/cm 2 ), followed by culturing for 40 min and then processing for immunoprecipitation). Lesser amounts of proteins were used in immunoprecipitation, compared with the above experiment. C, immunoprecipitation with a monoclonal antibody (Pab240) against p53 showed its interaction with MDM2 of 60 and 42 kDa, but not the full-length 90 kDa, in HCT116 cells. This population of p53 did not bind WOX1 effectively, suggesting that this p53 is a nonphosphorylated form. Pab240 recognizes conformationally altered p53 (49). Nutlin-3 induced activation of p53 and its binding with phosphorylated WOX1 but not with MDM2. D, HCT116 cells were pretreated with nutlin-3 (10 M) for 24 h and then exposed to CHX (300 M) for 1 h. Nutlin-3 prevented p53 from CHX-mediated degradation.   33 and their complex formation in the presence of MDM2. Nutlin-3 inhibits MDM2 and appears to alter p53 conformation (p53*), thereby stabilizing p53 and its interaction with WOX1. This p53 is not phosphorylated at key serines. UV light restores the complex formation of p-p53-p-WOX1-MDM2. B, WOX1 alone binds MDM2 probably via its C-terminal short-chain alcohol dehydrogenase/reductase domain, and nutlin-3 dissociates the binding. UV light restores the binding. staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, in the absence of functional WOX1, p53 protein expression is abolished, although p53 mRNA levels are not reduced. Time course analysis shows that the stability of ectopic wild type p53-DsRed is decreased in WOX1 knockdown cells. Interestingly, inhibition of MDM2 by nutlin-3 increases the binding of p53 and WOX1 and stability of p53. These observations suggest that WOX1 may act as a protein chaperone that stabilizes newly synthesized p53. Also, Tyr 33 -phosphorylated WOX1 stabilizes Ser 46phosphorylated p53 via direct binding interactions.
Recently, we have demonstrated that the protein levels of WOX1, isoform WOX2, and p-WOX1 are significantly down-regulated, along with increased phosphorylation of Tau, GSK-3␤, and extracellular signal-regulated kinase and neurofibrillary tangle formation, in the neurons of hippocampi of patients with Alzheimer disease (10). Remarkably, neuroblastoma SK-N-SH cells with WOX1 knockdown by siRNA have spontaneously induced Tau phosphorylation, enhanced phosphorylation of GSK-3␤, and extracellular signal-regulated kinase and enhanced tangle formation (10). Here, we found that WOX1 knockdown MDA-MB-231 cells have an increased ubiquitination of mutant p53. Indeed, the overall ubiquitination of cellular proteins is also increased in the absence of WOX1, as shown in MDA-MB-231 and other cells (data not shown). Apparently, WOX1 plays an important role in controlling protein stability.
p53 is consisted of an amino-terminal transactivation domain (two subdomains), a proline-rich (or growth-regulatory) region, a central zinc-binding core domain that binds DNA, and a nuclear localization and oligomerization domain at the carboxyl terminus (Fig. 2). MDM2 controls the nucleocytoplasmic trafficking of p53 in resting cells (52). MDM2 binds p53, and both undergo nuclear export to the cytosol, thereby resulting in proteasome-mediated degradation. The prolinerich region appears to provide stability of p53 in resisting MDM2-mediated degradation (53).
We show the likely presence of two forms of activated p53. Nutlin-3, a newly developed chemical (48), stabilizes and activates p53 without inducing phosphorylation on key serine residues (33). This activated p53 appears to be conformationally altered, since it can be recognized by a monoclonal antibody against conformationally altered p53 (Pab240) (49). Indeed, this p53 is present in low amounts in many types of cells expressing wild type p53 (data not shown). This p53 binds low molecular sizes of MDM2 of 60 and 42 kDa. Nutlin-3 dissociates the binding and appears to increase the levels of this conformationally altered p53. WOX1 stabilizes this nonphosphorylated p53 probably by binding to the proline-rich region. In contrast to the effect of nutlin-3, UV light and numerous apoptosis-inducing chemicals activate p53 via phosphorylation at Ser 46 and other serines. The phosphorylated p53 complexes with both MDM2 and WOX1. UV light appears to restore the formation of p53-MDM2-WOX1 complex in cells pretreated with nutlin-3, as determined by colocalization analysis.
In addition, we determined that WOX1 physically associates with MDM2 in p53-deficient H1299 cells. Similarly, UV light induces the binding of WOX1 with MDM2, whereas nutlin-3 does the opposite. Also, UV light restores colocalization of WOX1 with MDM2, suggesting an increased binding for both proteins. How MDM2 binds to WOX1 remains to be determined. Conceivably, the C-terminal shortchain alcohol dehydrogenase/reductase domain of WOX1 interacts with MDM2. This would allow binding of the N-terminal WW domain area of WOX1 with p53, thereby forming a trimolecular complex, WOX1-p53-MDM2, in cells such as COS7 fibroblasts and colorectal HCT116 cells.
MDM2 is involved in the regulatory control of nucleocytoplasmic trafficking of p53. As determined by immunofluorescence microscopy, we show that nutlin-3 increases nuclear localization of WOX1 and p53 in the nuclei but restricts MDM2 to the cytoplasm. It is likely that WOX1 increases p53 stability by enhancing the duration of p53 nuclear localization. UV light increases nuclear localization of WOX1, MDM2, and p53, along with their complex formation. We believe that WOX1 is likely to counteract with MDM2 in promoting nuclear export of p53.
Prolyl isomerase Pin1 possesses an N-terminal WW domain and binds to phosphorylated p53 on the Ser/Thr-Pro motifs, particularly at Ser 33 , Ser 315 , and Thr 81 , and the polyproline region (60,61). Ser 46 phosphorylation in p53 has no effect on Pin1 binding (60,61). The isomerase activity of Pin1 stabilizes and induces conformational changes of p53, thereby stimulating the DNA binding activity and transactivation function of p53 (60,61). Whether WOX1 stimulates p53-mediated DNA binding and transactivation function remains to be established.
We have shown that the non-ankyrin C terminus of IB␣ physically interacts with the proline-rich region and Ser 46 in p53. The binding could be abolished by deletion of Ser 46 or alteration of Ser 46 to Gly in p53 (29). However, alteration of Ser 46 to a conserved phosphorylation residue Thr still retains the binding interaction, suggesting that Ser 46 phosphorylation in p53 is involved in binding with IB␣. The p53-IB␣ complex dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-␤1-mediated growth suppression (29). Zhou et al. (34) confirmed our findings regarding p53 and IB␣ interactions. WW domain-containing proteins are known to bind proline-rich motifs, such as PPXY, PPLP, and others (62). However, phosphorylation of the conserved Tyr 33 in the first WW domain of WOX1 enables its interaction with a large context of proteins, independently of polyprolinebinding.Forexample,WOX1bindsJNK1inaTyr 33 phosphorylationdependent manner, whereas there is no apparent proline-rich motif in JNK1 (25). Similarly, Tyr 33 -phosphorylated WOX1 binds Smad4 in a Tyr 33 phosphorylation-dependent manner. 5 No proline-rich motifs are shown in Smad4. How Tyr 33 -phosphorylated WOX1 interacts with its binding partners remains to be established.
In summary, we have shown that in resting cells, a portion of cytosolic p53 complexes with MDM2 and WOX1. This binding is critical in controlling p53 activation, conformational changes, nuclear translocation, and ubiquitination/proteosomal degradation.