Oncogene (2008) 27, 5808–5820 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Recruitment of adenomatous polyposis coli and b-catenin to axin-puncta MC Faux, JL Coates, B Catimel, S Cody, AHA Clayton, MJ Layton and AW Burgess Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Victoria, Australia The adenomatous polyposis coli (APC)tumour suppressor coli (APC) form a complex that promotes the phos- is a multifunctional protein involved in the regulation of phorylation of b-catenin by glycogen synthase kinase-3-b Wnt signalling and cytoskeletal dynamics. Little is known (GSK3-b), which consequently targets b-catenin for about how APC controls these disparate functions. In this ubiquitination by bTrCP and destruction by the study, we have used APC- and axin-fluorescent fusion proteasome (Aberle et al., 1997; Orford et al., 1997; proteins to examine the interactions between these Kitagawa et al., 1999). Wnt stimulation and APC proteins and show that the functionally distinct popula- mutations disrupt the APC/axin complex and result in tions of APC are also spatially separate. Axin-RFP forms increased levels of b-catenin in the nucleus and cytoplasmic punctate structures, similar to endogenous cytoplasm (Munemitsu et al., 1995). b-Catenin is known axin puncta. Axin-RFP recruits b-catenin destruction to interact with members of the Tcf family of transcrip- complex proteins, including APC, b-catenin, glycogen tion factors to activate transcription of genes involved in synthase kinase-3-b (GSK3-b)and casein kinase-1- a proliferation and cellular transformation (Korinek (CK1-a). Recruitment into axin-RFP puncta sequesters et al., 1997). APC from clusters at cell extensions and this prevents its The function of APC in the regulation of the Wnt/ microtubule-associated functions. The interaction between b-catenin pathway has been studied in depth and the APC-GFP and axin-RFP within the cytoplasmic puncta is aberrant activation of b-catenin as a result of truncating direct and dramatically alters the dynamic properties of mutations in APC is recognized as a central event in APC-GFP. However, recruitment of APC to axin puncta colon cancer (Polakis, 1997, 2000; Nathke, 2004). is not absolutely required for b-catenin degradation. However, the spatial and temporal regulation of APC’s Instead, formation of axin puncta, mediated by the DIX interactions with Wnt signalling components and the domain, is required for b-catenin degradation. An cytoskeleton is less well understood. APC is localized at axinDDIX mutant did not form puncta, but still mediated the basal plasma membrane in association with micro- recruitment of destruction complex proteins and phos- tubules, to the peripheral ends of cell extensions (Nathke phorylation of b-catenin. We conclude that there are et al., 1996; Barth et al., 2002). This distribution of APC distinct pools of APC and that the formation of axin points to a major role for APC in cytoskeletal functions puncta, rather than the axin/APC complex, is essential associated with cell migration. APC binds to micro- for b-catenin destruction. tubules (Munemitsu et al., 1994; Su et al., 1995) and Oncogene (2008) 27, 5808–5820; doi:10.1038/onc.2008.205; regulates their growth and stability (Kita et al., 2006), published online 30 June 2008 and has been linked to the actin cytoskeleton via interactions with the Rac1/Cdc42 effector protein Keywords: APC; axin; b-catenin IQGAP1 (Watanabe et al., 2004), the Rac-specific exchange factor Asef (Kawasaki et al., 2000) and the Rho effector mDia (Wen et al., 2004). b-catenin has also recently been implicated in the clustering of APC at cellular protrusions (Sharma et al., 2006). However, it is Introduction not clear whether the ends of APC-loaded microtubules are the sites of the axin/b-catenin/GSK3-b/APC ‘de- Wnt signalling controls crucial stages in cell fate struction’ complex or whether this function takes place decisions, tissue development and maintenance and elsewhere in the cell. has been identified as a key signalling pathway in cancer The subcellular location of axin is less well defined (Logan and Nusse, 2004; Schneikert and Behrens, 2007). than APC. In Xenopus the physiological concentration When Wnt signalling is suppressed, the scaffold protein of axin is proposed to be exceedingly low (Lee et al., axin and the tumour suppressor adenomatous polyposis 2003), making detection at the subcellular level difficult. Endogenous axin has been detected in small, punctate Correspondence: Dr MC Faux, Epithelial Biology Laboratory, cytoplasmic structures that redistribute to large aggre- Ludwig Institute for Cancer Research, PO Box 2008, Royal gates close to the plasma membrane following treatment Melbourne Hospital, Parkville, Victoria 3050, Australia. with LiCl (which inhibits GSK3-b, mimicking Wnt E-mail: [email protected] Received 12 November 2007; revised 30 April 2008; accepted 30 May activation) (Levina et al., 2004; Wiechens et al., 2004). 2008; published online 30 June 2008 Ectopically expressed recombinant axin is localized in Recruitment of APC and b-catenin to axin puncta MC Faux et al 5809 cytoplasmic vesicles or puncta (Fagotto et al., 1999; destruction complex proteins and that formation of the Smalley et al., 1999; Schwarz-Romond et al., 2005). The axin puncta, rather than the axin/APC complex, is C-terminal DIX domain of axin is known to mediate critical for b-catenin degradation. self-association, which is thought to be important for the regulation of b-catenin (Hsu et al., 1999; Kishida et al., 1999; Sakanaka and Williams, 1999) and has recently been shown to associate into polymers that are Results required for puncta formation (Schwarz-Romond et al., 2007a, b). It has also been proposed that axin shuttles in To study the subcellular location and dynamic proper- and out of the nucleus (Wiechens et al., 2004) and that it ties of axin, we assessed the localization of both associates with microtubules and regulates their stability endogenous and recombinant axin fused to monomeric through dishevelled (Dvl) (Ciani et al., 2004). red fluorescent protein (RFP) (Figure 1a). Using a Although APC and axin have been shown to interact commercially available polyclonal axin antibody, we using immunoprecipitation (Hart et al., 1998), the observed endogenous axin in distinct puncta in the subcellular location of this interaction has not been well cytoplasm of Madin–Darby canine kidney (MDCK) defined. It is possible that only a small proportion of cells (Figures 1b and c). Both endogenous and APC associates with the axin complex. Recent biochem- recombinant axin puncta were evenly distributed ical data point to the existence of discrete populations of throughout the cytoplasm (Figures 1b–e), consistent APC where the APC/axin/b-catenin complex is separate with previous reports for endogenous axin in HEK293 from APC interactions with microtubules (Penman cells (Levina et al., 2004; Wiechens et al., 2004) and et al., 2005). These studies suggest that the spatial recombinant axin (Fagotto et al., 1999; Smalley et al., regulation of both APC and axin is a dynamic process 1999; Schwarz-Romond et al., 2005, 2007b). Axin-RFP but complex formation at the subcellular level has not was localized to cytoplasmic puncta when expressed been explored. In the present study we have used APC- in both MDCK (Figure 1e) and HEK293 T cells and axin- fluorescent fusion proteins to study axin (Supplementary Figures S1, S2). Similarly, epitope– regulation of APC subcellular localization and to tagged axin-HA (Figure 1d) formed cytoplasmic puncta, examine the dynamic interactions of these proteins in suggesting that the monomeric RFP tag did not affect its living mammalian cells. We examined whether other distribution. destruction complex proteins, b-catenin, GSK3-b and casein kinase-1-a (CK1a) were recruited to axin puncta Axin recruits APC to cytoplasmic puncta and whether axin puncta formation, mediated by the Endogenous APC concentrated in clusters at the ends of DIX domain of axin, is required for complex formation microtubules in subconfluent epithelial cells (Nathke and b-catenin degradation. Our data show that there are et al., 1996; Barth et al., 2002; Figure 2a). Transiently distinct subcellular pools of APC, that axin redistributes expressed APC-GFP was also concentrated in distinct APC from microtubule cell extensions to cytoplasmic clusters at the peripheral ends of microtubules in axin puncta, and that this prevents microtubule- MDCK cells (Figure 2b), mimicking the staining of associated functions of APC. We show that axin recruits endogenous APC in these cells (Figure 2a). In some Figure 1 Localization of axin to cytoplasmic puncta. (a) Schematic representation of domain structures of axin-, axinDRGS- and axinDDIX-RFP and APC-GFP fusion proteins. (b, c) Endogenous axin is localized to cytoplasmic puncta in Madin–Darby canine kidney (MDCK) cells. Cells were fixed and immunostained for endogenous axin with antibodies to axin-1. (c) High magnification of cytoplasmic puncta of endogenous axin. (d) MDCK cells transiently expressing axin-HA were fixed and immunostained with anti-HA antibodies. (e) MDCK cells expressing axin-RFP. Shown are single confocal sections. Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5810 cells, APC-GFP also decorated the microtubule network puncta affects microtubule-associated functions of APC. (see Figure 2f). In contrast, in cells expressing axin-RFP,