SHORT REPORTS The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library Tomohiro Kawaguchi 1,2,3 Shunsuke Kato 1,3 , Kazunori Otsuka 1 , Gou Watanabe 1 , Toshihiro Kumabe 2 , Teiji Tominaga 2 , Takashi Yoshimoto 2 and Chikashi Ishioka* ,1 1 Department of Clinical Oncology, Institute of Development, Aging and Cancer (IDAC), and Tohoku University Hospital, Tohoku University, Sendai 980-8575, Japan; 2 Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai 890-8574, Japan Tumor suppressor p53 forms a homo-tetramer through its COOH-terminal oligomerization domain and acts as a sequence-specific transcription factor. We have analysed the interrelation among the transcriptional activities, the structure and the cancer-related mutations in the oligo- merization domain by using a comprehensive missense mutation library. Here, we examined the ability of 184 mutant p53s in the domain to form an oligomer by expressing these mutant p53s in yeast, and compared the data with the previous information. We showed that specific residues in the a-helix and the b-strand of the oligomerization domain were critical for both oligomer formation and sequence-specific transactivation, and the activities were closely related. In particular, the a-helix was more sensitive to amino-acid substitutions than the b-strand. We found identity in the interrelation between the two activities, that is, monomer mutants were transcriptionally inactive whereas dimer and tetramer mutants retained their transcriptional activities. In TP53 mutation databases, a small number of mutations have been reported in this domain. Surprisingly, most do not encode p53s defective in functional properties. These results indicate that, although oligomer formation is essential for p53 transactivation function, the inactivation of oligomer formation and therefore the inactivation of transactivationmaynotbeessentialfortumorsuppression by p53 because they do not lead to oncogenic proteins. Oncogene (2005) 24, 6976–6981. doi:10.1038/sj.onc.1208839; published online 20 June 2005 Keywords: p53; oligomerization; transactivation p53 plays an important role to maintain genomic integrity from cellular stress by trans-activating the downstream genes involved in different cellular func- tions, such as cell cycle arrest (Slingerland and Bench- imol, 1991; el-Deiry et al., 1993; Tanaka et al., 2000) and apoptosis (Miyashita and Reed, 1995; Owen-Schaub et al., 1995; Polyak et al., 1997; Oda et al., 2000). Cellular stress, including DNA damage and hypoxia, stabilizes p53 protein by post-transcriptional modifica- tion, such as phosphorylation (Sakaguchi et al., 1998; Ashcroft et al., 1999), acetylation (Barlev et al., 2001) and prolyl isomerization (Zacchi et al., 2002; Zheng et al., 2002) in specific residues outside the central DNA- binding domain, converting the latent form of p53 into the active form. p53 acts as a tetramer through the carboxy-terminal oligomerization domain and several studies have shown the importance of this domain for p53 transcriptional activity, tumor suppressive activity and dominant negative effect of p53 (Shaulian et al., 1992, 1993; Unger et al., 1992; Halazonetis and Kandil, 1993). The structure of the oligomerization domain has been solved by both X-ray crystallography and NMR (Lee et al., 1994; Clore et al., 1995; Jeffrey et al., 1995; Miller et al., 1996; Chene et al., 1997; Mittl et al., 1998) and the structural information of specific residues in the solved ‘V-shaped’ structures is useful to better under- stand oligomerization. However, the contribution of each residue in oligomerization has not been validated biochemically. Previously, we have randomly mutagenized the domain by an error-prone PCR technique and yeast- based functional assay (Ishioka et al., 1995). We have isolated two interesting mutant p53s encoded by two missense mutations. The first mutant, L344P, identified as a loss of transactivation mutant, is located in the a-helix and had no ability to form dimers or tetramers (monomer mutant). The second mutant, K351E, identi- fied as a partial-transactivation mutant, has the ability to form dimers but not tetramers (dimer mutant) (Ishioka et al., 1997). L344P is not only defective in the in vitro experimental setting but also in the pathogenic setting because it has been reported as a causative germline mutation in a family with classic Li–Fraumeni syndrome (Varley et al., 1996). This mutant p53 is also shown to be a monomer mutant (Ishioka et al., 1997; Davison et al., 1998; Lomax et al., 1998). These results suggest that oligomer (either dimer or tetramer) formation is essential for the p53 transactiva- tion function and show that mutations defective in oligomer formation are as pathogenic as common Received 14 February 2005; revised 5 May 2005; accepted 6 May 2005; published online 20 June 2005 *Correspondence: C Ishioka, Department of Clinical Oncology, IDAC and Tohoku University Hospital, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; E-mail: chikashi@idac.tohoku.ac.jp 3 These authors contributed equally to this work Oncogene (2005) 24, 6976–6981 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc