Testing Models of the APC Tumor Suppressor/b-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in.80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene b-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity b-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all b-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that b-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all b-catenin-binding sites (including the 15Rs) and find that a direct b-catenin/APC interaction is also not essential for b-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby b-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock b-catenin in the destruction complex to increase the efficiency of b-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of b-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of b-catenin likely accounts for this difference. THE Wnt signaling pathway represents one of six evolutionary conserved pathways that collectively regulate animal development, yet are frequently misregulated in human disease (Clevers and Nusse 2012). During development, Wnt signaling regulates cell fate decisions that influence a myriad of developmental events as diverse as establishment of the vertebrate axis, control of bone development, and the wiring of the neural circuitry (Cadigan and Peifer 2009; Regard et al. 2012; Salinas 2012; Hikasa and Sokol 2013). Wnt signaling continues to be critical in adult homeostasis, as it is involved in the maintenance of certain mammalian stem cells (Holland et al. 2013). While Wnt signaling is clearly essential for an array of developmental events, misregulation of the pathway is the initiating event in the vast majority of colorectal cases. Truncating mutations in the negative regulator of Wnt signaling, adenomatous polyposis coli (APC), are responsible for both the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP) and .80% of spontaneous colon cancer cases (Polakis 2012). APC functions as a gatekeeper of the intestinal epithelium by mediating proteolytic degradation of the Wnt signaling effector b-catenin (bcat). At the molecular level, APC negatively regulates bcat by participating in a multiprotein “destruction complex” that consists of the core members Axin and the serine/threonine kinases glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1) (Stamos and Weis 2013). Once assembled into the destruction complex, bcat is sequentially phosphorylated by CK1 and GSK3, recognized Copyright © 2014 by the Genetics Society of America doi: 10.1534/genetics.114.166496 Manuscript received February 25, 2014; accepted for publication June 5, 2014; published Early Online June 13, 2014. Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.114.166496/-/DC1. These authors contributed equally to this work. Corresponding author: Department of Biology, P.O. Box 3003, Franklin & Marshall College, Lancaster, PA 17604. E-mail: [email protected] Genetics, Vol. 197, 1285–1302 August 2014 1285 by the SCFbTrCP E3 ubiquitin ligase, polyubiquitinated, and destroyed in the 26S proteasome. Thus in cells with an active destruction complex, bcat levels are low and Wnt target genes suppressed. In cells receiving Wnt signal, the Wnt ligand binds to a coreceptor complex consisting of the seven-pass transmembrane protein frizzled (Fz) and the single-pass membrane protein LRP5/6 (MacDonald and He 2012). Wnt binding to the receptor complex results in phosphorylation of the cytoplasmic tail of LRP5/6, which recruits Axin to the membrane. Membrane recruitment of Axin inactivates the destruction complex through a mechanism involving Disheveled (Dvl) (Bilic et al. 2007). He and colleagues suggest that Wnt signaling promotes an intramolecular conformational change in Axin that prevents assembly of bcat into the complex (Kim et al. 2013), whereas Clevers and colleagues propose that Wnt signaling prevents recruitment of the E3 ligase to an intact destruction complex (Li et al. 2012). In either case, bcat remains hypophosphorylated and escapes ubiquitination, and its intracellular protein levels rise. bcat then translocates to the nucleus and associates with TCF/ LEF proteins at Wnt-responsive elements (WREs) and displaces the Groucho repressor, thereby converting TCF/LEF from transcriptional repressors to activators. Ultimately these events result in expression of Wnt target genes such as c-myc and cyclin D1. While substantial experimental evidence supports this general model of Wnt signal transduction, several fundamental questions remain. One question is, What mechanistic role does APC play in the destruction complex? APC is a large protein with several putative protein–protein interaction domains; therefore, APC was initially thought to be the scaffold for the complex. However, Axin was subsequently identified as a negative regulator of Wnt signaling that binds all core components of the destruction complex (bcat, GSK3, CK1, APC, and Dvl), and other less wellcharacterized players such as the catalytic subunit of protein phosphatase 2A (PP2A) (Zeng et al. 1997; Sakanaka et al. 1998; Fagotto et al. 1999; Hsu et al. 1999). In addition, Axin can enhance the rate of bcat phosphorylation by CK1/GSK3, consistent with a scaffolding effect, whereas APC does not (Ha et al. 2004). These findings make Axin a stronger candidate to be the scaffold, leaving the mechanistic role of APC in the destruction complex mysterious. Recent biochemical and crystallographic data have suggested several models of APC function in the destruction complex, many of which have focused on the importance of bcat-binding sites on APC. APC contains two types of bcatbinding sites called 15-amino-acid repeats (15Rs) and 20amino-acid repeats (20Rs), with vertebrate APC containing four 15Rs and seven 20Rs (Figure 1A). Importantly, the 20Rs of APC are substrates for CK1/GSK3 phosphorylation (Ikeda et al. 2000; Ferrarese et al. 2007), whereas the 15Rs remain unphosphorylated. The affinity of each site for bcat was determined using isothermal calorimetry, which established that the 20Rs are higher-affinity bcat-binding sites than the 15Rs (Liu et al. 2006). In addition, phosphorylation of the 20Rs increases affinity for bcat up to 1500-fold. These findings prompted Kimmelman and Xu to propose a cycle of catalytic activity in the destruction complex in which the complex first assembles with bcat bound to Axin due to Axin’s single bcat-binding site possessing higher affinity for bcat than unphosphorylated 20Rs (Kimelman and Xu 2006; Xu and Kimelman 2007). Upon assembly, the 20Rs of APC are phosphorylated by CK1/GSK3, resulting in higheraffinity bcat-binding sites. bcat thus transfers from Axin to APC, presumably resulting in phosphorylated bcat being recognized by the SCFbTrCP E3 ligase. PP2A-mediated dephosphorylation of the 20Rs would serve to reset the cycle. Thus, the Kimmelman and Xu model would argue that at least some subset of 20Rs should be essential for APC function in the destruction complex. Liu and colleagues present an alternative view of APC’s function in the destruction complex, but maintain that phosphorylation of 20Rs should be essential (Su et al. 2008). They suggest that when bcat is phosphorylated by CK1/ GSK3, it is subject to rapid dephosphorylation by PP2A. APC serves to protect bcat from PP2A-mediated dephosphorylation, ensuring that phosphorylated bcat is recognized by the SCFbTrCP ligase and destroyed. The authors further demonstrate that a fragment of human APC encompassing the 20Rs was sufficient for this protective function, whereas the 15Rs of APC were nonprotective. Moreover, phosphorylation of APC was critical to prevent bcat dephosporylation, suggesting that CK1/GSK3 phosphorylation of the 20Rs mediates this effect. Surprisingly, APC binding to bcat prevents dephosphorylaton even though the APC-binding site on bcat does not overlap the bcat phosphodegron. Finally, Weis and colleagues suggest that the different bcat-binding sites on APC play an important role in finetuning bcat destruction in response to a gradient of Wnt signals (Ha et al. 2004). They propose that in the absence of Wnt signal, high-affinity bcat-binding sites (phosphorylated 20Rs) are important to maintain low levels of bcat, whereas low-affinity binding sites (15Rs) become critical in cells winding down from Wnt signal where bcat levels are relatively high. In such cells, the combination of lowand high-affinity bcat-binding sites could be important to rapidly destroy bcat and efficiently turn off Wnt signaling. In this study, we perform functional studies to test these models of APC function by generating APC transgenes lacking various bcat-binding sites. Vertebrate APCs are large proteins (approaching 300 kDa), thereby precluding the ability to reasonably perform structure/function studies in the context of a full-length protein. Instead, we use Drosophila as a model system. Flies and vertebrates have two highly conserved APC proteins (APC1 and APC2) that participate redundantly in Wnt regulation (Ahmed et al. 1998, 2002; Akong et al. 2002). APC1 and APC2 proteins share a conserved core region consisting of an N-terminal set of Armadillo repeats, a combination of 15Rs and 20Rs, and a series of SAMP motifs that bind the scaffold protein Axin (Figure 1A). 1286 R. J. Yamulla et al. In addition to this core region, APC1 proteins also contain a basic domain and an EB1 binding motif that are necessary for its localization to the plus end of microtubules. Prior studies have established that sequences C-terminal of the SAMP motifs are dispensable for proper Wnt regulation (Smits et al. 1999; Roberts et al. 2012b); thus we have selected Drosophila APC2 as a model to study APC function in the bcat destruction complex. Drosophila APC2 contains all components of the core region (including a set of Armadillo repeats, two 15Rs, five 20Rs, and two SAMP motifs) (McCartney et al. 1999; Yu et al. 1999); however, it is considerably shorter than other APC proteins, facilitating its use for structure/function studies. In designing Drosophila APC2 mutants devoid of particular bcat-binding sites, we were also informed by biochemical work demonstrating that the second 20R of human APC (20R2) lacks any detectable affinity for bcat, even when phosphorylated (Liu et al. 2006; Kohler et al. 2008). Initially, this finding suggested that 20R2 is a degenerate 20R that lost affinity for bcat through evolution; however, we recently showed that 20R2 is highly conserved in species as diverse as sea snails (Lottia gigantean), fruit flies, and humans (Roberts et al. 2011). Furthermore, we established that 20R2 is the only 20R individually required for APCmediated bcat destruction in both full-length Drosophila APC2 and fragments of human APC. Thus 20R2 has an essential role in mediating bcat destruction independent of the ability to directly bind bcat. Prior studies that sought to address the importance of high-affinity bcat-binding sites on APC for destruction complex function all eliminated 20R2 in addition to other combinations of 20Rs (Rubinfeld et al. 1997; Kunttas-Tatli et al. 2012). It is now clear that such APC mutants are defective in bcat destruction at least in part due to deleting or mutating 20R2 and thus may not accurately reflect the importance of direct bcat binding to APC. Therefore, in this study we test models of APC function by generating Drosophila APC2 transgenes lacking combinations of bcat-binding sites, but that keep 20R2 intact. We Figure 1 Schematics of human and Drosophila APCs. (A) Vertebrates and flies have two APC proteins that share a conserved core region consisting of Armadillo repeats, 15-amino-acid repeats (15Rs), 20-amino-acid repeats (20Rs), the catenin inhibitory domain (CID) (called region B in Drosophila), and SAMP motifs. The 15Rs and 20Rs both bind bcat, except 20R2. C-terminal sequences are more divergent, with some APCs containing a basic domain and an EB1-binding site. (B) Drosophila APC2 deletion constructs used in this study. b-Catenin Binding to APC 1287 perform functional studies with these transgenes in both human colon cancer cell lines and APC null Drosophila embryos, allowing us to assess which aspects of APC biology are conserved throughout evolution. Materials and Methods APC2 deletion mutants Drosophila APC2 deletion mutants were generated using the approach outlined previously (Roberts et al. 2011). Briefly, precise amino acid deletions were generated using a PCRsplicing approach, and the resulting PCR products were TOPO-TA cloned into the pCR8/GW/TOPO Gateway entry vector (Life Technologies). For cell culture experiments, APC2 entry vector constructs were gateway cloned (Life Technologies) into a modified ECFP-N1 destination vector (Clontech) containing the CMV promoter, an N-terminal EGFP tag, and a Gateway-3X STOP cassette. For transgenic fly lines, APC2 constructs were instead cloned into a modified pUAStattB vector (Basler laboratory, University of Zurich, Switzerland, GenBank accession no. EF362409) containing the endogenous APC2 promoter, an N-terminal EGFP tag, the Gateway-3X STOP cassette, and an attB site to facilitate targeted genomic integration using the PhiC31 approach. All transgenic flies were generated by BestGene (Chino Hills, CA), using the BL#9723 line, which results in transgene integration at cytogenetic position 28E7 on the second chromosome. The use of PhiC31 eliminates differences in expression levels caused by positional effects. All constructs were sequence verified. Yeast two-hybrid analysis Yeast two-hybrid (Y2H) analysis was performed using the Matchmaker System (Clontech). Briefly, the pGBKT7 and pGADT7 yeast vectors were engineered to be Gateway compatible by inserting a Gateway-3X STOP cassette downstream of the Gal4 DNA-binding domain or the Gal4 transcriptional activation domain, respectively. APC2 constructs were gateway cloned into the resulting pGBKT7-W, whereas full-length Armadillo was cloned into pGADT7-W. APC2 pGBKT7-W constructs were transformed into the Y2HGold yeast strain and Arm pGADT7-W into Y187, using the SC Easy Transformation kit (Life Technologies), and colonies were selected on –Trp or –Leu plates, respectively (Sigma Aldrich). Transformed yeast colonies were then mated in 23 YPAD media for 24 hr and plated on doubleselection –Leu –Trp plates. Yeast colonies were inoculated in liquid –Leu –Trp media, and b-galactosidase assays were performed using the Yeast b-galactosidase Assay Kit (Thermo Scientific; Pierce Chemical, Rockford, IL). Several different colonies were tested per experiment, and each experiment was independently conducted three times. Cell culture, transfections, and immunofluorescence SW480 cells were cultured at 37 and 5% CO2 in DMEM-H supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 13 Pen/Strep/Glutamine (GIBCO, Grand Island, NY). For transient transfections, SW480 cells were plated at a density of 2.5 3 105 cells per well in six-well plates and grown overnight. DNA constructs were then transfected using TurboFect (Thermo Fisher) according to the manufacturer’s instructions. For immunofluorescence studies, cells on coverslips were fixed 24 hr post-transfection with 4% formaldehyde in 13 phosphate-buffered saline (13 PBS) for 10 min. Cells were washed three times with 13 PBS, blocked for 15 min in 13 PBS containing 1% normal goat serum and 0.1% Triton X-100 (13 PBTN), and then antibody stained as previously described (Roberts et al. 2011). Primary antibodies were mouse anti-b-catenin (BD Transduction Laboratories, cat. no. 610153; 1:1000) and anti-Flag [Sigma (St. Louis), M2; 1:1000]. Secondary antibody was goat antimouse Alexa 568 (Life Technologies, 1:1000). To inhibit the proteasome, cells were treated with a cocktail of 25 mM MG132 and 25 mM ALLN for 6 hr prior to processing. TOP/FOP luciferase reporter assay The TOP/FOP Flash Luciferase constructs and the pRL Renilla transfection control were provided by Hans Clevers (Hubrecht Institute, Utrecht, The Netherlands). Luciferase assays were performed using the Dual Glo Luciferase System (Promega, Madison, WI) according to the manufacturer’s protocol. Briefly, SW480 cells were transiently cotransfected with 2 mg of the relevant APC2 construct, 1 mg of pRL, and 1 mg of either TOP or FOP Flash Luciferase reporter. After 24 hr, cells were lysed in a hypotonic 0.13 PBS solution and subjected to a 5-min freeze–thaw at 280 . Cells were scraped and cellular debris was pelleted in a microcentrifuge. Luciferase activity of each lysate was measured using a Perkin-Elmer (Norwalk, CT) EnSpire plate reader and normalized to Renilla signal. All samples were measured in triplicate per experiment, and three independent experiments were performed. None of the constructs displayed significant FOP flash activity. Quantifying bcat protein levels in transfected