Original Article

Oncogene (2010) 29, 5464–5474; doi:10.1038/onc.2010.275; published online 12 July 2010

PBK/TOPK interacts with the DBD domain of tumor suppressor p53 and modulates expression of transcriptional targets including p21

F Hu1, R B Gartenhaus1, D Eichberg1, Z Liu1, H-B Fang1 and A P Rapoport1

1Marlene and Stewart Greenebaum Cancer Center, University of Maryland, Baltimore, MD, USA

Correspondence: Dr AP Rapoport, Marlene and Stewart Greenebaum Cancer Center, University of Maryland, 22 South Greene Street, Baltimore, MD 21201, USA.
E-mail: arapoport@umm.edu

Received 29 September 2009; Revised 27 May 2010; Accepted 1 June 2010; Published online 12 July 2010.

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Abstract

PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that is overexpressed in a variety of tumor cells but its role in oncogenesis remains unclear. Here we show, by co-immunoprecipitation experiments and yeast two-hybrid analysis, that PBK/TOPK physically interacts with the tumor suppressor p53 through its DNA-binding (DBD) domain in HCT116 colorectal carcinoma cells that express wild-type p53. PBK also binds to p53 mutants carrying five common point mutations in the DBD domain. The PBK–p53 interaction appears to downmodulate p53 transactivation function as indicated by PBK/TOPK knockdown experiments, which show upregulated expression of the key p53 target gene and cyclin-dependent kinase inhibitor p21 in HCT116 cells, particularly after genotoxic damage from doxorubicin. Furthermore, stable PBK/TOPK knockdown cell lines (derived from HCT116 and MCF-7 cells) showed increased apoptosis, G2/M arrest and slower growth as compared to stable empty vector-transfected control cell lines. Gene microarray studies identified additional p53 target genes involved in apoptosis or cell cycling, which were differentially regulated by PBK knockdown. Together, these data suggest that increased levels of PBK/TOPK may contribute to tumor cell development and progression through suppression of p53 function and consequent reductions in the cell-cycle regulatory proteins such as p21. PBK/TOPK may therefore be a valid target for antineoplastic kinase inhibitors to sensitize tumor cells to chemotherapy-induced apoptosis and growth suppression.

Keywords:

PDZ-binding kinase; tumor suppressor p53; cyclin-dependent kinase inhibitor p21WAF1; PBK/TOPK

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Introduction

PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that was cloned as an interaction partner with the human tumor suppressor discs-large (hdlg) by yeast two-hybrid screening and as an IL-2 inducible gene in cytotoxic T lymphocytes (Abe et al., 2000; Gaudet et al., 2000). PBK/TOPK was also identified as a differentially expressed sequence tag in Burkitt's lymphoma cells (Simons-Evelyn et al., 2001). The murine homologue was cloned as an IL-6-stimulated gene from murine myeloma cells (Cote et al., 2002). PBK/TOPK mRNA is expressed in various tumor cell lines and in normal testicular and embryonic tissues (Simons-Evelyn et al., 2001). PBK/TOPK protein is also highly expressed in primary clinical samples from patients with aggressive hematopoietic neoplasms (Nandi et al., 2004).

PBK/TOPK functions partly as an MAP kinase kinase by phosphorylation of p38 mitogen-activated protein kinase (MAPK) (Abe et al., 2000; Nandi et al., 2004; Ayllón and O’Connor, 2007) and is active during mitosis through phosphorylation of residue Thr9 by cyclin B1/Cdk1 (Abe et al., 2000; Gaudet et al., 2000). During mitosis, PBK/TOPK forms a complex with cyclinB1/Cdk1 on microtubules that promotes cytokinesis through phosphorylation of PRC1 (Abe et al., 2007). Additional downstream targets of PBK/TOPK include the MAPKs JNK1 and ERK2, the latter forming a positive feedback loop with PBK/TOPK that may contribute to tumorigenesis in colorectal cancer cells (Oh et al., 2007; Zhu et al., 2007). Studies of neural progenitor cells indicate that phospho-PBK/TOPK is detected selectively in M-phase cells in association with condensed chromatin (Dougherty et al., 2005). Cell-cycle-specific regulation of PBK/TOPK is mediated partly by transcription factors E2F and CREB/ATF (Nandi and Rapoport, 2006). Previous studies also suggest a role for PBK/TOPK in cytokinesis and DNA damage sensing and repair through phosphorylation of histone H2AX (Matsumoto et al., 2004; Zykova et al., 2006; Ayllón and O’Connor, 2007).

These studies, which implicate PBK/TOPK in the regulation of mitosis and DNA repair, suggest that it may also have a significant role in cancer development and progression. Indeed PBK/TOPK is overexpressed in breast cancer, and siRNA-mediated reduction of PBK/TOPK expression suppresses breast cancer cell growth and clonogenicity (Park et al., 2006; Ayllón and O’Connor, 2007). In addition to its role in the MAPK oncogenic signaling pathway, PBK/TOPK has recently been found to be a downstream target of the EWS-FLI1 chimeric fusion protein that is present in 90% of cases of Ewing sarcoma (Herrero-Martin et al., 2009). We have previously shown that forced expression of a phosphomimetic mutant of PBK/TOPK resulted in loss of DNA damage checkpoint control leading to aneuploidy and suggested that this observation may be due to attenuation of p53 function (Nandi et al., 2007). In this study we present both biochemical and genetic evidence to show that PBK/TOPK (hereafter referred to as PBK) physically interacts with p53 through its DBD domain and downregulates its function as indicated by modulation of p53-associated targets including p21.

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Results

DNA-binding domain of p53 is required for physical interaction with PBK

In earlier work, using a yeast two-hybrid screen, we identified p53 as a potential interacting partner of PBK (Nandi et al., 2007). We sought to extend biological relevance to this finding and therefore examined the physical interaction between p53 and PBK under physiological conditions. Because both PBK and wild-type p53 are expressed in human colorectal carcinoma HCT116 cells, we used this cell line for co-immunoprecipitation (co-IP) assays and subsequent functional assays. Figure 1a shows that using either an antibody against PBK or an antibody against p53, it was possible to pull down both proteins in the co-IP experiments, whereas control normal serum IgGs did not pull down the protein complexes in either direction. Densitometry measurements found that about 3% of input p53 was pulled down by the PBK antibody, and 2.7% of input PBK was pulled down by the p53 antibody. These reciprocal co-IP experiments established that endogenous wild-type PBK and p53 physically interact in HCT116 cells.

Figure 1.
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PBK interacts with p53 through its DNA-binding domain (DBD). (a) Endogenous PBK and p53 interact in HCT116 cells. Western blot showing a reciprocal co-immunoprecipitation of endogenous PBK and p53 by either an antibody against PBK or an antibody against p53 but not control IgGs. The heavy chain (HC) of the antibody used for immunoprecipitation is seen in both the PBK lane and the IgG control lane of the p53 blot. (b) Diagram of EGFP-tagged full-length and deletion constructs of p53. (c) A western blot shows that strong protein expression was achieved for wild-type p53 and each of the p53 deletion mutants that were used for the co-IP mapping experiment. (d) Mapping region(s) of p53 that interact with PBK by examining interaction in extracts of co-transfected HCT116 cells. HCT116 cells were co-transfected with p3xflag-PBK and full-length or a deletion construct of p53. Western blot analysis of GFP immunoprecipitation showed that the DBD domain of p53 was necessary and sufficient to mediate protein–protein interaction between p53 and PBK.

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To identify the domain on p53 responsible for this interaction, we transfected HCT116 cells with p3xflag-PBK in combination with a plasmid encoding either the full-length or one of a series of deletion mutants of p53 and performed co-IP assays (Figure 1b). Figure 1d shows that full-length p53 and all p53 mutants that contained the DBD domain could co-immunoprecipitate with PBK, but EGFP-p53-mt3, the p53 mutant in which the DBD domain was deleted, did not co-immunoprecipitate with PBK. However, EGFP-p53-DBD, which contained only the DBD domain of p53, co-immunoprecipitated with PBK (Figure 1d). Western blot analysis also showed that all the p53 deletion mutants were strongly expressed at the protein level (Figure 1c). These data suggested that the DBD domain of p53 was both necessary and sufficient to mediate the protein–protein interaction between p53 and PBK in HCT116 cells.

To confirm these co-IP results, we performed a yeast two-hybrid assay using bait plasmid pGBKT7-PBK and prey constructs expressing either the full-length or the same deletion mutants of p53 that were used in the interaction domain mapping studies by co-IP. As shown in Figure 2a, colonies grew from transformants of pGBKT7-PBK and all p53 prey constructs that contained the DBD domain of p53. No colonies grew from cells co-transformed with pGBKT7-PBK and pGADT7-p53-mt3, in which the DBD domain was deleted. Thus, the yeast two-hybrid experiments confirmed that the DBD domain of p53 was responsible for mediating the p53–PBK interaction.

Figure 2.
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(a) The DNA-binding domain (DBD) of p53 mediates strong interaction between p53 and PBK in yeast two-hybrid assay. Bait construct pGBKT7-PBK and prey construct expressing full-length or deletion mutants were co-transformed into AH109 strain yeast cells, transformants were plated on -His/-Leu/-Trp triple dropout plates and incubated at 25°C for 7 days. Colonies appeared wherever a prey construct that contained the DBD domain of p53 was co-transformed into the host cells. Control experiments co-transforming yeast cells with empty bait vector pGBKT7 and each of the prey constructs were performed to exclude false-positive interactions. All p53 fragments contained in the prey constructs were the same as those contained in the mammalian expression constructs used for the co-IP experiments. (b) PBK inhibits p53-mediated transcription in a dose-dependent manner. HCT116 cells grown in 12-well plates were co-transfected with 0.5μg pEGFP-p53-wt or pEGFP-N1 vector in combination with 0.5μg pp53-TA-luc and increasing amount of p3xflag-PBK as indicated. p3xflag-cmv-7.1 plasmid was used to balance total amount of DNA transfected in each well. After 24h, cell extracts were prepared and luciferase activities were quantified. In all the experiments, firefly luciferase activity was normalized against total protein in the same sample and expressed as mean±s.d. of triplicates from a representative experiment. (c) Cell extract from a parallel transfection with panel (b) was blotted for analysis of PBK and p53 expression; β-actin was blotted for loading control.

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Because p53 mutations occur more frequently in the DBD domain than any other domain, we investigated whether PBK has relevance to mutation-promoted tumorigenesis by checking whether DBD mutations of p53 could affect PBK binding. We introduced mutations into the prey construct pGADT7-p53-DBD at five ‘hotspot’ positions (R175H, R248W, R273H, G245S and R282W), and conducted yeast two-hybrid experiments with bait construct pGBKT7-PBK (Soussi et al, 2006). As shown in Supplementary Figure S1, none of these mutations abolished binding of PBK.

PBK inhibited p53-mediated transcription in HCT116 cells

To begin to clarify the functional significance of the molecular interactions between PBK and p53, we first examined the effect of PBK on p53 activity in reporter gene assays. The p53-mediated transcriptional response was measured by using pp53-TA-luc, in which the expression of the luciferase gene was mediated by two p53-binding fragments (Kern et al., 1991; Komarova et al., 1997). As shown in Figure 2b, although overexpression of PBK alone had little effect on expression of luciferase activity from this reporter construct when promoted by basal levels of endogenous p53, forced expression of PBK appeared to suppress the transcription induced by ectopically expressed p53 in a dose-dependent manner.

PBK knockdown activates p21 expression in HCT116 cells

Because the cyclin-dependent kinase inhibitor p21 is a well-known transcriptional target of p53, we investigated whether PBK could also affect transcription of p21 through its interaction with p53. For these studies, we used a luciferase construct pp21-luc, in which the luciferase gene is controlled by a 2.4kb DNA fragment that includes the human p21 promoter (el-Deiry et al., 1993). As shown in Figure 3b, transient PBK knockdown using three PBK-specific RNAis increased the p21 promoter activity by about 3- to 4-fold (P<0.01). Using HCT116 cells that were transiently transfected in parallel to those depicted in panel b, we also showed a 2- to 3-fold increase in p21 mRNA expression by qPCR (panel 3c). Western blotting confirmed that the three PBK RNAis (RNAi1, RNAi2 and RNAi3) all suppressed PBK protein expression (panel 3d). To show specificity of this PBK RNAi effect, we used RNAi2 to study whether knockdown of PBK expression would stimulate the promoter function of two genes that are unrelated to the p53 pathway (STAT3 and DR1) and found no effect (panels 3e and f). To further test the effect of PBK knockdown on the expression of p21 at the protein level, we conducted immunoblotting assays using the stable PBK knockdown cell line HCT116-shPBK1 and the control cell line HCT116-RS (Figure 3a). In the PBK knockdown cells, the p21 level was significantly elevated compared with that in the control cell line. However, the level of p53 protein expression did not change, suggesting that modulation of p53 activity by PBK, rather than a change in p53 level, mainly affected the expression of p21. To further investigate whether p53 is involved in the activation of p21 by PBK knockdown, we treated both cell lines with doxorubicin, a strong p53 activator. The data in Figure 3a show that, in the presence of 1μM doxorubicin, p53 levels increased similarly in both cell lines. However, the p21 level was further increased in the PBK knockdown cells compared with the wild-type HCT116 cells. These data are consistent with a model of PBK inhibition of p53 function.

Figure 3.
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PBK knockdown specifically increases p21 expression. (a) PBK knockdown cell line HCT116-shPBK and control wild-type cell line HCT116-RS were treated with 1μM doxorubicin or DMSO for 24h. Cell extracts were blotted for protein levels of PBK, p53 and p21. β-Actin was blotted for loading control. (b) PBK-specific RNAis-activated transcription from pp21-luc in which luciferase gene was controlled by a 2.4kb fragment of p21 promoter. HCT116 cells maintained in 12-well plates were transfected with 10pmol PBK-RNAi or scrambled control RNAi in combination with 0.5μg pp21-luc. Cell extracts were measured for luciferase activity 48h after transfection. Data were normalized against the total protein in each sample. Average and standard deviation from three transfections are shown. (c) PBK-specific RNAis activated endogenous p21 gene transcription. Cellular RNAs were extracted from parallel transfections with those in panel b and subjected to reverse transcription quantitative PCR analysis for p21 expression. (d) Cell extracts from parallel transfections with those in panel b were collected and blotted to check PBK knockdown. β-Actin was blotted for loading control. (e) pstat3-luc and pDR1-luc were not activated by PBK knockdown in luciferase assays conducted in parallel with those for pp21-luc by following the same protocol as in panel b, except that only RNAi2 was used. (f) Cell extracts from parallel transfections with those in panel e were collected and blotted to check PBK knockdown. β-Actin was blotted for loading control.

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PBK modulation of p21 expression is dependent on p53

To show that the observed modulation of p21 expression by PBK was dependent on p53 function, we examined the effect of PBK knockdown (using RNAi2) on p21 expression in the presence and absence of p53. For these studies, we obtained stable lines derived from HCT116 cells that carried homozygous (HCT116 p53−/−) or heterozygous (HCT116 p53+/−) deletions of the p53 gene. As shown in Figure 4a, PBK knockdown had no effect on p21 reporter activity in the HCT116 p53−/− cell line whereas p21 reporter activity significantly increased in the p53+/+ line as observed before. The heterozygous HCT116 p53+/− cell line appeared to show an intermediate effect. A similar pattern was observed when p21 mRNA expression was measured by qPCR. As shown in Figure 4b, PBK knockdown had no effect on the residual p21 mRNA level in the HCT116 p53−/− cells, induced a minor increase in p21 mRNA expression in the HCT116 p53+/− cells and nearly doubled the level of p21 mRNA expression by qPCR in the HCT116 p53+/+ cells. One possible mechanism whereby PBK could downregulate p53 transcriptional function would be interference with p53-DNA binding. To test this possibility, we performed a chromatin immunoprecipitation (ChIP) assay was performed. As shown in Figure 4d, overexpression of PBK decreased recruitment of p53 by the p21 promoter in HCT116 cells that were transiently transfected with p3xflag-PBK.

Figure 4.
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PBK modulation of p21 expression is dependent on p53. (a) Cellular p53 level influenced PBK knockdown-induced transcription from pp21-luc. HCT116 p53+/+, p53+/− and p53−/− cells grown in 12-well plates were transfected with 10pmol of PBK RNAi2 or scramble control RNAi in combination with 0.5μg pp21-luc. Cell extracts were measured for luciferase activity 48h after transfection. Data were normalized against the total protein in each sample. Average and standard deviation from three transfections are shown. (b) Cellular p53 level determined the effect of PBK knockdown on endogenous p21 gene transcription. Cellular RNAs were extracted from parallel transfections with those in panel a and subjected to reverse transcription quantitative PCR analysis for p21 expression. (c) Cell extracts from parallel transfections with those in panels a and b were collected and blotted to check p53 content and PBK knockdown. β-Actin was blotted for loading control. (d) PBK decreased recruitment of p53 by p21 promoter in ChIP analysis. HCT116 cells were plated into 100mm cell culture Petri dish to 80% confluency, the following day cells were transfected with 20μg p3xflag-PBK or empty vector. After 24h chromatin was collected and immunoprecipitated with anti-p53 or nonspecific rabbit IgG antibodies, and p21 promoter in the inputs and immunoprecipitates was detected by 30 cycles of PCR. Overexpression of PBK was confirmed by western blot analysis of cell extracts from parallel transfections. β-Actin was blotted for loading control.

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Phenotypic studies of PBK knockdown cell lines

Having shown in HCT116 cells that PBK knockdown could upregulate p53 function and p21 expression, we hypothesized that stable PBK knockdown cell lines would show predictable phenotypic changes regarding proliferation, cell-cycle progression and sensitivity to apoptosis. First, we compared the cell growth curve of the PBK knockdown stable cell line HCT116-shPBK1 to the growth of control HCT116-RS cells. As expected, HCT116-shPBK1 cells showed a significantly elongated population doubling time (P<0.0001) and slower growth (Figures 5a–c). Second, cell-cycle progression was examined after treatment of both the stable PBK knockdown HCT116-shPBK1 cell line and the parental HCT116 cell line with the genotoxin doxorubicin or dimethyl sulfoxide (DMSO). As shown in Figure 6a, in the PBK knockdown cell line HCT116-shPBK1 the %G2/M cells increased from 10.5 to 54.8% after exposure to 1μM doxorubicin. The %G2/M cells increased from 16 to 34% in the presence of DMSO diluent alone, suggesting that PBK knockdown could lead to cell-cycle arrest in the absence of genotoxins. Similar results were obtained after treatment of a second stable PBK knockdown cell line derived from MCF-7 breast cancer cells (Supplementary Figure S2). A separate HCT116-shPBK2 stable cell line derived using a second shRNA construct also showed accumulation of G2/M cells when compared with HCT116-V-RS cells carrying a scrambled, nonspecific shRNA construct (Supplementary Figure S3). These phenotypic changes correlated to DMSO/doxorubicin-induced increases in p53 protein level, as shown by western blotting, that were exaggerated in the PBK knockdown lines as compared with the control cell lines (Figure 6c). Finally, as shown in Figures 6d and e, apoptosis assayed by Trypan blue staining was significantly increased in both the HCT116 and MCF-7 stable PBK knockdown cell lines as compared with the control cell lines after exposure to either DMSO or doxorubicin. Similar results were obtained by Annexin V staining (data not shown).

Figure 5.
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PBK knockdown significantly elongates doubling time in HCT116 cells. (a) A total of 2 × 104 cells were plated on 60mm cell culture dish; cell number was counted using a hemocytometer every 24h for 5 days. (b) Plot of linear regression (blue line/triangles: HCT116-RS cells; red line/circles: HCT116-shPBK1 cells). (c) Data are shown as average doubling time and standard deviation from four parallel experiments. (d) Western blot analysis showed PBK knockdown in HCT116-shPBK1 cells. β-Actin was blotted for loading control.

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Figure 6.
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Phenotypic changes in stable PBK knockdown cell lines regarding cell-cycle distribution and apoptosis. Representative results from three independent experiments looking at the effects of PBK knockdown on cell-cycle distribution are shown. (a, b) A total of 2 × 106 HCT116-RS and HCT116-shPBK1 cells were plated in a 60mm cell culture dish. The following day cells were treated with DMSO or 1μM doxorubicin for 24h, collected, stained with PI and subjected to FCM analysis for cell-cycle distribution. A significant increase in %G2/M cells is apparent for the HCT116-shPBK1 stable knockdown cell line under both the conditions. (c) Cells treated in parallel with those in panels a and b and Supplementary Figure S2 were lysed and subjected to western blot analysis for expression of PBK and p53. β-Actin was blotted as a loading control. (d) A total of 1 × 106 cells from the HCT116-RS and HCT116-shPBK1 stable lines, and (e) 1 × 106 MCF-7-RS and MCF-7-shPBK1 cells were plated in six-well plates. Cells were serum starved for 24h the following day. The medium was then changed to medium that contained 10% fetal bovine serum and 1μM doxorubicin or solvent DMSO. After 24h, cells were collected and stained with Trypan blue. Live and dead cells were counted using a hemocytometer. Average and s.d. from three independent experiments are shown, indicating that for both the HCT116-shPBK1 and MCF-7-shPBK1 stable cell lines, PBK knockdown augments apoptosis. (f) MCF-7 cells treated in parallel with those in panels c and e were lysed and subjected to western blot assay for PBK and p53 to show downregulation of PBK. β-Actin was blotted for loading control. A comparable blot for the stable HCT116-shPBK1 cell line is depicted in Figure 5d.

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Gene microarray studies

To identify other genes that might be targets of PBK, we treated HCT116-RS and HCT116-shPBK1 cells with 1μM doxorubicin for 24h. Cellular RNA was extracted from cells and subjected to microarray analysis using the Affymetrix (Santa Clara, CA, USA) GeneChip Human Gene 1.0 ST Array. Relative to the empty-vector cell line, the stable knockdown cell line showed about a 2.6-fold decrease in PBK mRNA. Expression levels of at least nine p53 target genes involved in cell-cycle regulation and/or apoptosis were also significantly altered by PBK knockdown. Among these were p21 (Bunz et al., 1998; Wendt et al., 2006), DUSP1 (Li et al., 2003), THBS1 (Guo et al., 1997; Jimenez et al., 2000; Harada et al., 2003), NOXA (Oda et al., 2000), BAK (Pohl et al., 1999), FAS (Müller et al., 1998; Pohl et al., 1999), CASP10 (Rikhof et al., 2003) (all upregulated), and Bcl-xL (Bartke et al., 2001), which was downregulated. G2E3 (Brooks et al., 2007, 2008), a newly characterized gene involved in G2/M cell-cycle progression, was found to be suppressed by PBK knockdown, but its association to p53 is unknown. We validated the microarray analysis results for all nine genes by qRT–PCR (see Table 1 and Supplementary Figure S4).


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Discussion

Several lines of evidence suggest that the serine-threonine kinase PBK may contribute to tumor development and growth. Among these is that PBK was cloned on the basis of a yeast two-hybrid screen of HeLa cells using the hDlg tumor suppressor protein as bait (Gaudet et al., 2000). The hDlg tumor suppressor, the human homologue of the Drosophila Discs-large protein, interacts with multiple oncogenic proteins including the adenomatous polyposis coli tumor suppressor protein, the human papillomavirus E6 transforming protein and the adenovirus E4 transforming protein 9ORF1 (Matsumine et al., 1996; Kiyono et al., 1997; Lee et al., 1997).

In this study we show that PBK also interacts with p53, another key tumor suppressor protein. Our model system was HCT116 human colorectal cancer cells as this line is known to express wild-type p53 and therefore could provide more physiologic conditions for studying protein–protein interactions involving p53. Using both reciprocal co-IP experiments of unmodified PBK and p53 and yeast two-hybrid analysis, we showed that PBK physically interacts with p53 specifically through its DNA-binding domain (DBD). The DBD or central domain of p53 contains about 80% of the p53 mutations that occur in cancer cells (Soussi, 2007). It is also the critical interaction site for several important proteins that regulate p53 function including the ASPP proteins which shift p53 function toward apoptosis induction through upregulation of proapoptotic proteins Bax, PUMA and PIG3 (Patel et al., 2008). The antiapoptotic proteins BCL-xL and Bcl2 also interact with p53 through binding to the DBD domain with Bcl2 binding exclusively to wild-type p53 (Tomita et al., 2006; Sot et al., 2007).

To test whether PBK binding to wild-type p53 could modulate its transcriptional function, we studied the effect of PBK on expression of the cell-cycle inhibitor p21, a key transcriptional target for p53. Using transient co-transfection assays in HCT116 cells, we observed that PBK knockdown by three PBK-RNAis led to a significant increase in luciferase activity from a p21 promoter-luciferase construct. In addition, PBK knockdown by RNAi led to a significant increase in p21 mRNA by quantitative PCR analysis. A similar result was obtained at the p21 protein level using the stable PBK knockdown cell line HCT116-shPBK1. Under conditions of genotoxic damage by doxorubicin, p21 levels increased in the stable PBK knockdown cells (HCT116-shPBK1) in response to p53 induction, to a much greater degree than in the stable empty-vector control cell line (HCT116-RS). Knockdown of PBK expression had no effect on the promoter functions of two genes that are not known to be part of the p53 pathways (STAT3 and DR1). These data suggest that PBK inhibits p53 transactivation of p21 expression, which is partially released by PBK knockdown. Furthermore, overexpression of PBK led to interference with p53 binding to the p21 promoter region by ChIP assays. These data are consistent with the model that PBK interacts with the DBD of p53 and thereby inhibits p53 binding to target gene promoter sequences.

Because of the high prevalence of p53 mutations in cancer, we also studied whether PBK could bind to certain p53 tumor mutants. These Y2H studies showed that PBK binding was retained. The functional consequences of this interaction could be different than the functional consequences of PBK binding to wild-type p53. Indeed, BclxL binds to both wild-type and mutant p53 proteins (although with reduced affinity) yet only the interaction with wild-type p53 results in the conformational change in BclxL, which leads to proapoptotic activity (Hagn et al., 2010). In addition, mutant p53 proteins show gain-of-function properties including association with the nuclear matrix, formation of transcriptionally active protein complexes and sequestration of proapoptotic proteins (reviewed in Strano et al., 2007). Through binding to p53 mutants, PBK could modulate these oncogenic functions.

To strengthen the model that PBK modulation of p21 expression depends on interaction with p53, we examined the effect of PBK knockdown in HCT116 cells lines that were heterozygous or homozygous for p53 gene deletions. These studies confirmed that PBK knockdown had no effect on p21 expression in the p53 knockout line (HCT116-p53−/−), significantly increased p21 expression in the wild-type (p53+/+) line and showed an intermediate response in the heterozygous line (p53+/−) perhaps reflecting a p53 dose-dependent effect.

These findings suggest that PBK overexpression may promote tumor cell survival and resistance to chemotherapy-induced apoptosis through suppression of p21 expression and p53 function. One prediction of this model is that PBK knockdown in tumor cells should decrease proliferation and tumorigenicity through increased p21 expression and p53 function. Indeed three stable PBK knockdown cell lines showed accumulation in the G2/M phase of the cell cycle and increased apoptosis after exposure to DMSO or doxorubicin when compared with control lines that carried empty vectors or nonspecific shRNAs.

These findings complement earlier work showing that ectopic expression of a phosphomimetic mutant of PBK in HT1080 fibrosarcoma cells (known to carry wild-type p53 genes) attenuates G2/M checkpoint control during doxorubicin treatment (Nandi et al., 2007). Our findings also agree with previous studies showing that reduced PBK expression in MCF-7 breast cancer cells led to slower growth and reduced clonogenicity in soft agar assays (Ayllón and O’Connor, 2007). These investigators also showed that MCF-7 tumor cells with knocked down PBK expression exhibited decreased survival following DNA damage from UV or chemotherapeutic agents as well as impaired generation of phosphorylated H2AX (γ-H2AX), an early response to double-strand DNA breaks (Ayllón and O’Connor, 2007). Other investigators showed that knocked down expression of PBK in HCT116 colorectal carcinoma cells decreased tumorigenicity (Zhu et al., 2007).

The gene microarray studies reinforced the functional relationship between PBK and p53 by identifying at least seven p53 target genes (in addition to p21) involved in regulation of cell cycle and apoptosis and whose expression levels were modulated by PBK knockdown.

This article adds to the growing recognition that PBK is an oncoprotein and should be considered a valid target for the development of novel antineoplastic therapeutics. Furthermore, based on its restricted pattern of expression to tumor cells, testis, embryonic tissues and adult progenitor cells (Simons-Evelyn et al., 2001; Dougherty et al., 2005; Park et al., 2006), PBK may be considered a novel cancer/testis antigen that may also have a role in maintenance of tumor stem cells. Future studies are warranted to identify small molecular inhibitors of PBK and test them for the ability to sensitize tumor cells to chemotherapy-induced apoptosis and growth suppression.

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Materials and methods

Antibodies

Primary antibodies: rabbit anti-PBK, rabbit anti-p53 and a mouse monoclonal antibody against p21 were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-β-actin, rabbit anti-GFP polyclonal antibodies and anti-FLAG M2 monoclonal antibody were purchased from Sigma-Aldrich (St Louis, MO, USA). Secondary antibodies: both horseradish-peroxidase-conjugated donkey anti-rabbit IgG and horseradish-peroxidase-conjugated sheep anti-mouse IgG were purchased from GE Healthcare Amersham (Piscataway, NJ, USA).

Cloning vectors

The mammalian expression vector p3xflag-cmv-7.1 was purchased from Sigma-Aldrich. The mammalian expression vector pEGFP-N1 and yeast two-hybrid vectors pGBKT7 and pGADT7 were purchased from Clontech (Mountain View, CA, USA).

Gene microarray studies

To identify other genes and pathways that might be targets of PBK, we treated HCT116-RS and HCT116-shPBK cells with 1μM doxorubicin for 24h. Cellular RNA was extracted from cells and subjected to microarray analysis. Labeled cRNA was hybridized onto Affymetrix GeneChip Human Gene 1.0 ST Array. Further details on microarray procedures and analysis can be found in Supplementary Methods.

Plasmid constructions

For PCR reactions and DNA cloning, a high-fidelity DNA polymerase enzyme Platinum Taq DNA Polymerase High Fidelity and 100mM dNTP set were used (Invitrogen, San Diego, CA, USA). Restriction enzymes BamHI, HindIII, NheI, KpnI and a DNA ligase kit Quick Ligation Kit were purchased from New England Biolabs (Ipswich, MA, USA).

Details of specific plasmid constructions are provided in Supplementary Methods.

The reporter construct pp53-TA-luc, in which luciferase expression is under control of two p53-binding fragments, was purchased from Clontech. The p21 promoter reporter construct pp21-luc was a generous gift from Bert Vogelstein at Johns Hopkins University School of Medicine.

Cell lines, cell culture and transfection studies

The HCT116 human colon carcinoma cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum (Quality Biological Inc., Gaithersburg, MD, USA), and 1% penicillin/streptomycin. Cell lines HCT116-p53−/− and HCT116-p53+/− were generously provided by Dr Vogelstein. MCF-7 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. New stable cell lines obtained after transfection were maintained in the same media supplemented with 2μg/ml puromycin. RNAis and plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen). PBK Validated Stealth RNAi, Stealth RNAi Negative Control, Dnase I and recombinant RNAseOUT were purchased from Invitrogen. Doxorubicin was purchased from Sigma-Aldrich.

Immunoprecipitation studies

For co-IP of endogenous PBK and p53, HCT116 cells grown in a 100mm Petri dish were lysed in standard radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Details of the immunoprecipitation are provided in Supplementary Methods. The immunoprecipitates were eluted by the 2 × SDS sample buffer and 20% of eluate was resolved by SDS–polyacrylamide gel electrophoresis for western blot analysis.

To identify the p53 domain(s) responsible for protein–protein interaction between PBK and p53 by co-IP, we generated a mammalian expression construct pEGFP-p53-wt in which full-length p53 cDNA was fused to the N terminus of an EGFP tag. In addition, using PCR, we generated a series of GFP constructs each containing a p53 deletion mutant lacking one or more functional domains. Co-transfections were performed using a plasmid p3xflag-PBK, which encodes a Flag-tagged PBK (see Supplementary Methods for details).

Yeast two-hybrid assay

All reagents for the Y2H assay were purchased from Clontech. The assay was performed in accordance with the manufacturer's instructions. The AH109 yeast strain was used for yeast two-hybrid analysis. Bait plasmid pGBKT7-PBK (1μg) together with prey plasmid (1μg) were used to transform competent AH109 cells. Transformants were streaked on -His/-Leu/-Trp triple dropout plates and incubated at 25°C for 7 days (see Supplementary Methods for details).

Quantitative RT–PCR

HCT116 cells were transfected with PBK Validated Stealth RNAis and Scrambled RNAi Negative Control. Total cellular RNAs were extracted 48h after transfection using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNAs were reverse transcribed into cDNA with the Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer's protocol. Quantitative RT–PCR analysis of p21 gene expression was carried out using QuantiTect SYBR Green RT–PCR Kit from Qiagen on an ABI 7900HT Fast Real-Time PCR System, according to the manufacturer's instructions (see Supplementary Methods for primer details). To verify the results of the qPCR measurements, we run the PCR products on 3% agarose gels and sequenced after purification.

Western blot analysis

For western blot analysis, 20μg of protein was fractionated on NuPAGE 4–12% Bis-Tris Gels (Invitrogen), and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% fat-free milk for 1h at room temperature and then probed with 1:1000 dilution of primary antibody overnight at 4°C, followed by incubation with 1:10000 dilution of horseradish-peroxidase-linked secondary antibody. The antibody–antigen complex was visualized by ECL chemiluminescence (GE Healthcare Amersham) and developed on Kodak Biomax Light Film (Sigma-Aldrich). Membranes were reblotted with anti-β-actin antibody to control for uneven protein loading.

Luciferase assay

HCT116 cells were seeded in 12-well plates and grown to 80% confluence. On the following day, cells were transfected with plasmids as indicated. In each case, correspondent empty vectors were used to balance the amount of plasmids used in each transfection. Cell lysates were collected 48h after transfection. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) on a Mithras LB 940 Multimode Reader (Berthold Technologies, Wildbad, Germany). The protein content in each sample was measured and used to normalize the luciferase activity.

Establishment of PBK knockdown stable cell lines

To establish the stable PBK knockdown cell lines HCT116-shPBK1, HCT116-shPBK2 and MCF-7-shPBK1, we transfected 5μg pRS-shPBK1 or pRS-shPBK2 into HCT116 or MCF-7 cells with the Lipofectamine 2000 transfection reagent. Starting 24h after transfection, cells were selected in 2μg/ml puromycin (Sigma-Aldrich) for 3 weeks. Control cell lines HCT116-RS, HCT116-V-RS and MCF-7-RS were established by transfecting cells with 5μg pRS-vector or pGFP-V-RS that expresses a 29-mer scrambled shRNA and selected under the same conditions. Puromycin-resistant cells were assayed for protein expression by immunoblotting with a monoclonal antibody against PBK (Cell Signaling Technology).

Cell growth studies using PBK knockdown stable cell line HCT116-shPBK1

A total of 20000 wild-type HCT116-RS cells or PBK knockdown HCT116-shPBK1 cells were seeded on each 60mm dish at day 0. Cells in four dishes of each cell line were counted using a hemocytometer every 24h for 5 days.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assay was performed using the EZ ChIP kit (Millipore, Billerica, MA, USA). Briefly, HCT116 cells were lysed, sonicated and exposed to rabbit anti-p53 antibody (Cell Signaling Technology) or the same amount of control IgG. Details of the procedure are provided in Supplementary Methods.

Flow cytometry analysis

For fluorescence-activated cell sorting analysis, collected cells were fixed in 80% ice-cold ethanol for 1h. Cells were then rehydrated in phosphate-buffered saline, treated with the propidium iodide staining solution (50mg/ml propidium iodide and 0.5mg/ml RNaseA in phosphate-buffered saline) for 30min at room temperature and analyzed using an FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical analysis

Data are presented as means±s.d. The Student's t-test was used for statistical analysis, with P<0.05 defined as statistically significant. Longitudinal regression analysis was conducted to compare the growth rates between the wild-type and PBK knockdown cells.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was supported in part by philanthropic gifts from Mr Willard Hackerman, Mr and Mrs Robert Becker, and Mr and Mrs Gaylord Christle. Additional funds were provided by the Marlene and Stewart Greenebaum Cancer Center.

Supplementary Information accompanies the paper on the Oncogene website