First published online 7 October 2008
doi: 10.1242/jcs.026351
Journal of Cell Science 121, 3541-3552 (2008)
Published by The Company of Biologists 2008
Identification of ANKRD11 as a p53 coactivator
Paul M. Neilsen1,*,
Kelly M. Cheney1,
Chia-Wei Li2,
J. Don Chen2,
Jacqueline E. Cawrse1,
Renée B. Schulz1,
Jason A. Powell3,
Raman Kumar1 and
David F. Callen1
1 Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Discipline of Medicine, University of Adelaide and Hanson Institute, IMVS, Adelaide, SA 5000, Australia
2 Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA
3 Cytokine Receptor Laboratory, Department of Human Immunology, Hanson Institute, IMVS, Adelaide, SA 5000, Australia
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Fig. 1. ANKRD11 associates with p14ARF, p53 and PML in extranucleolar inclusions. (A) GFP-ANKRD11 localizes to subnuclear domains that are visible using phase-contrast microscopy (upper panels). MCF-10A cells were transduced with a recombinant retrovirus expressing GFP-ANKRD11, selected in geneticin and imaged using confocal microscopy. MCF-10A cells stably expressing GFP-ANKRD11 were immunostained with anti-nucleophosmin antibody (nucleolar marker; red) and DAPI (4',6-diamidino-2-phenylindole; DNA; blue) (lower panels). (B) Endogenous ANKRD11 colocalizes with p53. MCF-10A cells were either untreated or treated with mitomycin C (20 µg/ml) for 6 hours and immunostained with anti-ANKRD11 and anti-p53 (Ab-5) antibodies. The relative p53 protein levels in these cells with up to 6 hours of mitomycin C treatment was determined by western blot analysis using an anti-p53 antibody. β-actin was used as a loading control. (C) ANKRD11-myc localized exclusively to p14ARF-positive extranucleolar inclusions. HEK293T cells were transiently transfected with constructs expressing GFP-p14ARF and ANKRD11-myc. GFP-p14ARF localized to both nucleoli (hollow arrowheads) and extranucleolar inclusions (filled arrowheads). Localization of ANKRD11-myc was determined through immunostaining with anti-myc antibody. (D) Endogenous PML colocalizes with p14ARF and ANKRD11-myc in extranucleolar inclusions. HEK293T cells from C, ectopically expressing GFP-p14ARF and ANKRD11-myc, were immunostained with anti-myc and anti-PML antibodies.
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Fig. 2. ANKRD11 interacts with p53 in vivo and in vitro via the ANK domain. (A) ANKRD11 coimmunoprecipitates with endogenous p53. Protein complexes from either MCF-7 (retroviral-mediated GFP-ANKRD11 expression; mitomycin C treated for 6 hours) or HEK293T cell lysates (endogenous ANKRD11) were immunoprecipitated using an anti-ANKRD11 antibody or pre-immune serum. Immunoprecipitated protein complexes were analyzed by western blot analysis using the indicated antibodies. WB, western blot; IP, immunoprecipitation. (B) p53 interacts with the N-terminal region of ANKRD11. HEK293T cells were transiently transfected with constructs expressing one of four different Flag-tagged regions of the ANKRD11 protein or empty pCMV-Tag2 vector as indicated. Protein complexes from total cell lysates were immunoprecipitated using anti-Flag antibody coated Sepharose beads. Inputs (lanes 1-5) or immunoprecipitates (lanes 6-10) were detected by western blot analysis. Low levels of non-specific p53 binding to the Sepharose beads were also detected (lanes 7-10). (C) ANKRD11 interacts with endogenous p53 through the ankyrin repeat domain. HCT116 cells treated with mitomycin C (6 hours) or HEK293T cells were transiently transfected with constructs encoding myc- or Flag-tagged ANKRD11144-313aa protein (ANK domain) or empty vector, respectively. Protein complexes were immunoprecipitated using the appropriate antibodies and western blotted. (D) ANKRD11 directly interacts with p53 through the ankyrin repeat domain. Purified recombinant GST (lanes 9 and 11) or GST-ANKRD11144-313aa (lanes 10 and 12) fusion proteins were incubated with either MBP (lanes 9 and 10) or MBP-p53 (lanes 11 and 12) amylose beads. GST (lane 7) and GST-ANKRD11144-313aa (lane 8) inputs and interacting protein complexes (lanes 9-12) were western blotted using an anti-GST antibody. To confirm their intactness, all proteins were resolved by SDS-PAGE and visualized by colloidal Coomassie Blue staining (lanes 1-6).
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Fig. 3. ANKRD11 expression enhances p53 transcriptional activity. Saos-2 (left) or HeLa (right) cells were cotransfected with p53-responsive reporter constructs encoding 17 tandem p53-REs (left), the CDKN1A promoter region (middle) or the MDM2 promoter region (right), together with pRL-TK and constructs expressing myc-p53 and ANKRD11-myc as indicated. Empty pLNCX2 vector was added to equalize the total amounts of plasmid used in various treatments. In HeLa cells, ANKRD11 expression resulted in a minimal increase in reporter activity in the absence of exogenous myc-p53. This is presumably due to ANKRD11-mediated activation of endogenous p53 in HeLa cells, because this phenomenon was not observed in the p53-null Saos-2 cell line. Data are represented as mean ± s.e.m. of triplicates. The expression of protein levels from HeLa cells transiently transfected with the CDKN1A promoter region reporter, pRL-TK, myc-p53 and ANKRD11-myc constructs as indicated was determined by western blot analysis (inset).
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Fig. 4. ANKRD11 regulates p53-mediated expression of p21waf1 in breast cell lines. (A) ANKRD11 expression in the indicated breast cell lines was determined by real-time RT-PCR. Cultures of MCF-7, MB-468 or MB-231 stably expressing GFP-ANKRD11 (MCF-7-ANK-1 to ANK-3; MB-468-ANK-1; MB-231-ANK-1 to ANK-3) or negative control cell lines (MCF-7; MB-468; MB-231) were established from single colonies after retroviral transduction and selected in geneticin. Total ANKRD11 expression (endogenous plus exogenous; light shading) in these stably expressing cell lines were determined and compared with the levels of endogenous ANKRD11 expression (dark shading) in the indicated breast cell lines. The p53 status and 16q LOH status of each breast cell line is indicated. (B) Restoration of ANKRD11 expression increases p21waf1 expression only in MCF-7 and MB-468 cell lines. Both CDKN1A mRNA and p21waf1 protein levels (inset) were determined in cultures stably expressing GFP-ANKRD11 or the negative control cell cultures as described in A using real-time RT-PCR analysis and western blot analysis. (C) ANKRD11 upregulates p21waf1 expression in a p53-dependent manner. MCF-7 or MCF-7-ANK-1 cells as described in A were transfected with either p53-specific or scrambled siRNA for 72 hours. Both p53 and p21waf1 protein levels were determined using western blot analysis, and p21waf1 levels were quantified by densitometry (representative of two independent experiments). (D) Silencing of ANKRD11 by shRNA reduces p53-mediated transcription. MCF-10A cultures stably expressing ANKRD11 shRNA (MCF-10A-shANK) show a reduction in both mRNA and protein levels of ANKRD11 compared with MCF-10A cells stably expressing scrambled shRNA (MCF-10A-SCR). MCF-10A-shANK and MCF-10A-SCR cells were either untreated or treated with mitomycin C for 12 hours and the relative CDKN1A mRNA levels and p53 protein levels were determined using real-time RT-PCR analysis and western blot analysis, respectively.
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Fig. 5. ANKRD11 associates with hADA3 and P/CAF to enhance the acetylation and DNA-binding activity of both wild-type p53 and mutant p53R273H. (A) Yeast two-hybrid assay showing interaction between ANKRD112369-2663aa and hADA3 (left panel). Yeast cells coexpressing either pGBT-ANKRD112369-2663aa and empty pACT2 vector, pACT-hADA3 and empty pGBT9 vector, or pACT-hADA3 and pGBT-RAC3-N showed no activation of the β-galactosidase reporter. ANKRD11 interacts with hADA3 in vitro (centre panel). GST-pull down assay shows that GST-ANKRD112369-2663aa but not GST alone pulled down significant amounts of 35S-labeled hADA3. All proteins were resolved by SDS-PAGE and visualized by Coomassie Blue staining to confirm they were intact. ANKRD11 nuclear foci colocalize with P/CAF (right panel). ANKRD11 nuclear foci colocalize with P/CAF. COS-7 cells were transiently transfected with Flag-tagged P/CAF and full-length HA-tagged ANKRD11. Transfected cells were immunostained with anti-HA (ANKRD11; green) and anti-Flag (P/CAF; red) antibodies and DAPI (DNA; blue). (B) Restoration of expression of ANKRD11 increases p53 acetylation at Lys320. Total p53 and acetyl-lysine p53 (Lys320) protein levels were detected in MCF-7, MB-468 and MB-231 cultures stably expressing GFP-ANKRD11 or the negative control cell cultures previously described (Fig. 4A) through western blot analysis using the appropriate antibodies. MCF-7-ANK-1 was cultured in the presence or absence of mitomycin C for 6 hours and is presented as a representative MCF-7 derivative from (Fig. 4A). (C) The p53 coactivator function of ANKRD11 is dependent upon Lys320. H1299 cells were cotransfected with the p53-responsive reporter constructs encoding the CDKN1A promoter region, together with pRL-TK and constructs expressing either myc-p53, myc-p53-K320R or ANKRD11-myc as indicated. Empty pLNCX2 vector was added to equalize the total amounts of plasmid used in various treatments. Data are represented as mean ± s.e.m. of triplicates. The expression of protein levels from H1299 cells transiently transfected with either myc-p53 or myc-p53-K320R constructs was determined by western blot analysis (inset). (D) ANKRD11 enhances the DNA-binding activity of both wild-type p53 and mutant p53R273H. Lysates from either MCF-7-ANK-1 (upper panels) or MB-468-ANK-1 (lower panel) cells stably expressing GFP-ANKRD11 or the negative control MCF-7 or MB-468 cells (described in Fig. 4A) were promoter precipitated using either beads charged with the CDKN1A promoter region or uncharged beads. Inputs or promoter precipitates were analyzed by western blot using anti-p53 (detecting wild-type and p53R273H) and anti-acetyl-p53 (Lys320) antibodies, as indicated.
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Fig. 6. ANKRD11 is a p53 target gene. (A) p53 transcriptionally activates a reporter gene driven by an ANKRD11 intronic sequence carrying a p53-RE. HeLa cells were cotransfected with a construct expressing myc-p53 and the reporter constructs pGL3-Basic (left; empty vector), pGL3-Basic-ANKRD11-p53-RE-Luc (middle) or pGL3-Basic-ANKRD11-p53-RE-mut-Luc (right). The relative luciferase activity was determined in cell lysates as described in Fig. 3. Data are represented as mean ± s.e.m. of triplicates. The expression level of the myc-p53 construct compared with endogenous p53 levels in HeLa cells was determined by western blot analysis (inset). (B) ANKRD11 expression correlates with increasing p53 protein levels during the DNA damage response. ANKRD11 expression in either HCT116 or HCT116 (TP53–/–) cells was determined at time-points 0, 8, 24 and 48 hours post-treatment with mitomycin C. The expression of ANKRD11 in HCT116 (wild-type p53) cells was normalized to that observed in the isogenic HCT116 (TP53–/–) derivative to monitor only the p53-dependent modulation of ANKRD11 expression during the DNA damage response. Induction of p53 protein levels following treatment with mitomycin C were determined by western blot analysis (inset). (C) HCT116 (p53–/–) cells were transfected in a 24-well format with the indicated amounts of myc-p53 (A), and the relative ANKRD11 mRNA levels in these treatments were determined using real-time RT-PCR analysis.
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© The Company of Biologists Ltd 2008
