3541 Research Article Introduction The p53 protein is the most important tumor suppressor in the cell and is described as ‘the guardian of the genome’ (Lane, 1992). The function of p53 is predominantly dependent on its activity as a sequence-specific transcription factor, controlling the expression of various target genes that mediate biological functions such as cell- cycle arrest, apoptosis, senescence, differentiation, DNA repair and inhibition of angiogenesis and metastasis (Liu and Chen, 2006). Approximately 50% of all cancers contain a mutation in the TP53 gene (Vogelstein et al., 2000). The six most frequently occurring tumorigenic mutations of p53 cluster within the DNA-binding surface, involving residues that either directly contact DNA (‘DNA- contact’ mutations – residues R248 and R273) or perturb the structural integrity of the DNA-binding surface of p53 (‘structural’ mutations – residues R175, G245, R249 and R282) (Bullock and Fersht, 2001). DNA-contact mutants generally retain a similar structural integrity and thermodynamic stability to that displayed by wild-type p53; however, the mutation might significantly impair or even completely abrogate its ability to interact with its cognate DNA-response element (Bullock et al., 2000). Rescue of DNA- contact mutants has been demonstrated through restoration of their DNA-binding affinities following treatment with a p53-derived C- terminal peptide (Selivanova et al., 1997), small p53-activating molecules (CP-257042, CP-31398 and PRIMA-1) (Bykov et al., 2002; Foster et al., 1999) or the introduction of second site suppressor mutations (Brachmann et al., 1998). Despite these recent advances, the development of successful pharmacological therapeutics designed to rescue p53 DNA-contact mutants has proven to be a challenging task because of the complex biomechanistics that occur during the restoration of mutant p53 function. The p53 transcription factor is stabilized and activated in response to various forms of intracellular or extracellular stress. Stabilization and activation of p53 occurs through a complex pattern of post- translational modifications, including phosphorylation and acetylation. The acetylation of specific lysine residues of p53 is mediated by several acetyltransferases, including p300/CBP (Lys164, Lys370, Lys372, Lys373, Lys381, Lys382 and Lys386), Tip60/hMOF (Lys120) and P/CAF (Lys320) (Gu and Roeder, 1997; Sakaguchi et al., 1998; Sykes et al., 2006; Tang et al., 2008). Earlier studies have shown that acetylation of p53 promotes its ability to bind its cognate DNA-response element in vitro (Gu and Roeder, 1997; Sakaguchi et al., 1998); however, these findings are not consistent with recent in vivo data (Liu and Chen, 2006; Tang et al., 2008; Toledo and Wahl, 2006). It has since been shown that the critical function of acetylation in vivo is not to enhance the DNA- binding affinity of p53, but rather to enhance its interaction with coactivators or histone acetyltransferases (HATs) (Barlev et al., 2001), or perturb its interaction with Mdm2 (Tang et al., 2008) at the promoters of p53-responsive genes. Recent evidence also suggests that acetylation of specific lysine residues in p53 may influence the selection of specific p53 target genes, resulting in induction of either growth arrest or apoptotic pathways (Di Stefano et al., 2005). hADA3 (alteration/deficiency in activation) has been identified as a novel p53-interacting protein capable of recruiting p300/CBP and P/CAF to p53 (Wang et al., 2001). Ectopic expression The ability of p53 to act as a transcription factor is critical for its function as a tumor suppressor. Ankyrin repeat domain 11, ANKRD11 (also known as ANR11 or ANCO1), was found to be a novel p53-interacting protein that enhanced the transcriptional activity of p53. ANKRD11 expression was shown to be downregulated in breast cancer cell lines. Restoration of ANKRD11 expression in MCF-7 (wild-type p53) and MDA-MB- 468 (p53 R273H mutant) cells suppressed their proliferative and clonogenic properties through enhancement of CDKN1A (p21 waf1 /CIP1) expression. ShRNA-mediated silencing of ANKRD11 expression reduced the ability of p53 to activate CDKN1A expression. ANKRD11 was shown to associate with the p53 acetyltransferases and cofactors, P/CAF and hADA3. Exogenous ANKRD11 expression enhanced the levels of acetylated p53 in both MCF-7 and MDA-MB-468 cells. ANKRD11 enhanced the DNA-binding properties of mutant p53 R273H to the CDKN1A promoter, suggesting that ANKRD11 can mediate the restoration of normal p53 function in some cancer-related p53 mutations. In addition, ANKRD11 itself was found to be a novel p53 target gene. These findings demonstrate a role for ANKRD11 as a p53 coactivator and suggest the involvement of ANKRD11 in a regulatory feedback loop with p53. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/21/3541/DC1 Key words: Acetylation, Ankyrin repeat domain protein, Breast cancer, p53, p53 mutant rescue Summary Identification of ANKRD11 as a p53 coactivator Paul M. Neilsen 1, *, Kelly M. Cheney 1 , Chia-Wei Li 2 , J. Don Chen 2 , Jacqueline E. Cawrse 1 , Renée B. Schulz 1 , Jason A. Powell 3 , Raman Kumar 1 and David F. Callen 1 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 *Author for correspondence (e-mail: Paul.Neilsen@imvs.sa.gov.au) Accepted 29 July 2008 Journal of Cell Science 121, 3541-3552 Published by The Company of Biologists 2008 doi:10.1242/jcs.026351 Journal of Cell Science JCS ePress online publication date 7 October 2008