Oncogene (2010) 29, 1663–1671 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Contribution of the 15 amino acid repeats of truncated APC to b-catenin degradation and selection of APC mutations in colorectal tumours from FAP patients EM Kohler1, K Brauburger, J Behrens and J Schneikert Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen-Nu¨rnberg, Erlangen, Bayern, Germany The adenomatous polyposis coli (APC) protein is a leads to the development of polyps in the gastrointest- negative regulator of the mitogenic transcription factor inal tract that ultimately evolve as aggressive and fatal b-catenin by stimulating its proteasomal degradation. tumours in affected people. Besides gastric, duodenal This involves several APC domains, including the binding and colorectal cancer, familial adenomatous polyposis sites for axin/conductin, the recently described b-Catenin coli (FAP) patients may also develop desmoid tumours Inhibitory Domain (CID) and the third 20 amino acid (Kotiligam et al., 2008). repeat (20R3) that is a b-catenin-binding site. The four 15 Many functions have been attributed to adenomatous amino acid repeats (15R) and the 20R1 are also b-catenin- polyposis coli (APC) (Nathke, 2006; Akiyama and binding sites, but their role in b-catenin degradation Kawasaki 2006; Aoki and Taketo 2007; Schneikert has remained unclear. We show here that binding of and Behrens, 2007a; McCartney and Nathke, 2008), but b-catenin to the 15R of APC is necessary and sufficient to it is best understood as a component of the canonical target b-catenin for degradation whereas binding to the wnt signalling pathway, controlling the fate of the 20R1 is neither necessary nor sufficient. The first 15R transcription factor b-catenin (Clarke, 2006; Clevers, displays the highest affinity for b-catenin in the 15R-20R1 2006; Taketo, 2006; Polakis, 2007; Fodde and Brabletz module. Biallelic mutations of the APC gene lead to 2007). In the presence of a proliferative signal provided colon cancer in familial adenomatous polyposis coli by an extracellular ligand from the wnt family, cytosolic (FAP) and result in the synthesis of truncated products b-catenin accumulates, translocates into the nucleus and lacking domains involved in b-catenin degradation but still stimulates the transcription of target genes (Behrens having a minimal length. The analysis of the distribution et al., 1996). Thereby, b-catenin translates the wnt signal of truncating mutations along the APC sequence in colo- into changes in the transcriptional state that ultimately rectal tumours from FAP patients revealed that the first manifest by cell proliferation. In the absence of a wnt 15R is one target of the positive selection of mutations signal, b-catenin is destroyed in the proteasome where that lead to tumour development. it is conveyed only after a critical phosphorylation Oncogene (2010) 29, 1663–1671; doi:10.1038/onc.2009.447; step catalysed in the so-called degradation complex published online 7 December 2009 (Kimelman and Xu, 2006). In this complex, axin/ conductin recruits the kinases glycogen synthase kinase Keywords: adenomatous polyposis coli; APC; b-catenin; 3b and casein kinase 1 that sequentially phosphorylate colon cancer; mutation b-catenin (Liu et al., 2002). This process is drastically enhanced by the presence of APC (Munemitsu et al., 1995) that displays several domains involved in b- catenin degradation (Figure 1). The SAMP repeats Introduction constitute binding sites for axin/conductin (Behrens et al., 1998). The seven 20 amino acid repeats (20R) are Familial adenomatous polyposis coli is an autosomal b-catenin-binding sites (Rubinfeld et al., 1993). Among dominant genetic disorder characterized by the germline the latter, a role in b-catenin degradation has been transmission of a mutation in the adenomatous poly- demonstrated in detail for the third 20R (20R3) only posis coli gene (Groden et al., 1991, 1993; Olschwang (Rubinfeld et al., 1997; Kohler et al., 2008). In contrast, et al., 1993). Somatic inactivation of the remaining allele the 20R2 cannot bind to b-catenin (Liu et al., 2006; Kohler et al., 2008). Phosphorylation of the 20R1 and the 20R3 increases their affinity for b-catenin (Tick- Correspondence: Dr J Schneikert, Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nu¨rnberg, Glu¨ckstrasse 6, enbrock et al., 2003; Ha et al., 2004; Xing et al., 2004; Erlangen, Bayern, 91054, Germany. Liu et al., 2006; Ferrarese et al., 2007). The b-Catenin E-mail: [email protected] Inhibitory Domain (CID) domain is located between the 1Current address: Department of Psychiatry and Psychotherapy, 20R2 and the 20R3 and is essential in truncated APC to University of Erlangen-Nu¨rnberg, Schwabachanlage, Erlangen, Germany. target b-catenin for degradation (Kohler et al., 2009). Received 15 June 2009; revised 23 October 2009; accepted 3 November The four 15 amino acid repeats (15R-A to D) are also b- 2009; published online 7 December 2009 catenin-binding sites (Su et al., 1993; Eklof Spink et al., Functional analysis of adenomatous polyposis coli EM Kohler et al 1664 Figure 1 Degradation of b-catenin can be independent of its interaction with APC. SW480 cells were transiently transfected with the indicated truncated APC expression constructs, encoding products fused to YFP at their N terminus. The CID domain, the 15 amino acid-, 20 amino acid- and SAMP repeats are shown as boxes. A black, M-labelled box indicates a mutated repeat that cannot bind to b-catenin, except the 20R3 for which the affinity is only decreased, although drastically. Definitive inactivation of the 20R3 requires additional mutations, which is indicated by MM (Kohler et al., 2008). Flag, empty control vector. To analyse the transcriptional activity of b-catenin, cells were co-transfected with either a b-catenin-dependent luciferase reporter gene (TOPglow) or a control reporter gene lacking functional b-catenin-binding sites (FOPglow), together with a b-galactosidase expression vector as an internal control to correct for variations in the transfection efficiency. Cells were harvested 24–36 h post transfection and cell extracts were submitted either to western blotting (WB) using either an anti-GFP or an anti-b-actin antibody, or to a luciferase assay (TOPglow/ FOPglow). Data are represented as the ratio of TOPglow to FOPglow values corrected by the b-galactosidase values and are the mean ( þ /À s.d.) of three independent experiments. In parallel, transfected cells were fixed and stained with an anti-b-catenin antibody. The column on the right (IF) indicates for each YFP–APC construct the percentage of transfected cells negative for b-catenin. At least hundred cells were scored for each construct. The intracellular localization of the YFP–APC constructs is also shown. 2001; Liu et al., 2006), but it is not known whether they 2009). These animals do not develop tumours in the have a role in b-catenin degradation. gastrointestinal tract. In some cases however, the CID APC mutations as found in the germline of FAP domain that allows truncated APC to target b-catenin patients lead to premature termination of translation for degradation even in the absence of the 20R3 and the and therefore to the synthesis of truncated products SAMP repeats in the appropriate cell line, can be (Miyoshi et al., 1992; Powell et al., 1992; Miyaki et al., retained in a colorectal tumour if additional molecular 1994, 2008; Segditsas and Tomlinson, 2006). Mutations events lead to its inactivation (Kohler et al., 2009). Thus, of both APC alleles in FAP tumours are interdependent, APC truncated shortly after the CID can be used that is, the site of the germline mutation determines the experimentally to target efficiently b-catenin for degra- nature and the location of the somatic mutation dation if it is expressed in a permissive cell line. (Lamlum et al., 1999; Rowan et al., 2000; Albuquerque Despite the negative selection, colon cancer cells et al., 2002; Crabtree et al., 2003). This results in a always retain a truncated APC of a minimal length. In complex distribution of APC mutations along the 95% of the cases the fragment extends up to at least the sequence, which is partially explained by the necessity end of the 20R1 (Kohler et al., 2008). At the molecular of removing or inactivating important domains involved level, the reasons of this positive selection are not in the degradation of b-catenin (that is, the SAMP known, but its strength indicates that the retained frag- repeats, the 20R3 and the CID domain). As a conse- ment probably fulfils an essential function. Actually, quence, b-catenin is constitutively stabilized. The SAMP RNA interference experiments have shown that APC repeats and the 20R3 are almost systematically excluded truncated shortly after the 20R1 and lacking the CID from truncated APC (Kohler et al., 2008). The necessity domain, is required for optimal cell proliferation of removing both domains is illustrated by the avail- (Schneikert and Behrens, 2006) and can still exert a ability of mice heterozygous for APC isoforms truncated control on the transcriptional activity of b-catenin either shortly after the first SAMP repeat (APC þ /1638T (Schneikert et al., 2007b). This is in line with the recent mice) (Smits et al., 1999) or between the 20R3 and analysis of APC þ /1322T mice (Pollard et al., 2009) the first SAMP repeat (APC þ /1572T mice) (Gaspar et al., carrying an APC truncation falling shortly after the Oncogene Functional analysis of adenomatous polyposis coli EM Kohler et al 1665 20R1. These mice display a more severe polyposis but for degradation (Kohler et al., 2009). All transfected polyps with lower amounts of nuclear b-catenin than in constructs were expressed at a similar level and mice, in which truncated APC does not contain any displayed an identical diffuse and cytoplasmic intracel- b-catenin-binding site.