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• W3192327720 abstract "•β-Catenin destruction complex function recapitulated with purified proteins•AXIN1 polymers and APC promote β-catenin capture, phosphorylation, and ubiquitylation•Oncogenic APC truncation mutants are hypomorphs promoting β-catenin recruitment•APC directly binds the ubiquitylation machinery The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin’s phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin’s flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFβ-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling. The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin’s phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin’s flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFβ-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling. The highly conserved Wnt/β-catenin signaling pathway coordinates key events in early embryogenesis, tissue homeostasis, and regeneration, governing stem cell maintenance, cell fate specification, and cell proliferation (Clevers et al., 2014Clevers H. Loh K.M. Nusse R. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control.Science. 2014; 346: 1248012Crossref PubMed Scopus (779) Google Scholar; Steinhart and Angers, 2018Steinhart Z. Angers S. Wnt signaling in development and tissue homeostasis.Development. 2018; 145: dev146589Crossref PubMed Scopus (201) Google Scholar). It is one of the most frequently mutated pathways in cancer (Sanchez-Vega et al., 2018Sanchez-Vega F. Mina M. Armenia J. Chatila W.K. Luna A. La K.C. Dimitriadoy S. Liu D.L. Kantheti H.S. Saghafinia S. et al.Cancer Genome Atlas Research NetworkOncogenic signaling pathways in The Cancer Genome Atlas.Cell. 2018; 173: 321-337.e10Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar). The degree of its activation is the outcome of balanced activities of two opposing multi-protein complexes that determine the fate of newly synthesized cytoplasmic β-catenin: the β-catenin destruction complex (DC) and the Wnt signalosome (Gammons and Bienz, 2018Gammons M. Bienz M. Multiprotein complexes governing Wnt signal transduction.Curr. Opin. Cell Biol. 2018; 51: 42-49Crossref PubMed Scopus (99) Google Scholar; Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref PubMed Scopus (621) Google Scholar; van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar). The DC predominates at basal Wnt/β-catenin signaling and, via phosphorylation-dependent ubiquitylation, earmarks β-catenin for proteasomal degradation. Receptor engagement by Wnt growth factors converts the DC into a receptor-associated Wnt signalosome complex, where β-catenin phosphorylation and ubiquitylation are attenuated, resulting in increased levels and nuclear accumulation of β-catenin and the expression of β-catenin/T cell factor/lymphoid enhancer-binding factor (TCF/LEF) target genes (Gammons and Bienz, 2018Gammons M. Bienz M. Multiprotein complexes governing Wnt signal transduction.Curr. Opin. Cell Biol. 2018; 51: 42-49Crossref PubMed Scopus (99) Google Scholar). In the DC, the scaffolding proteins and tumor suppressors axis inhibition protein 1 (AXIN1) and adenomatous polyposis coli (APC) collaborate to co-recruit the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β) jointly with their substrate β-catenin (Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref PubMed Scopus (621) Google Scholar; van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar; Figure 1A). This enables the sequential phosphorylation of an N-terminal β-catenin phosphodegron and the subsequent ubiquitylation of newly synthesized β-catenin by a SKP1-CUL1-F box (SCF) E3 ubiquitin ligase complex containing the substrate recruitment component β-TrCP (SCFβ-TrCP) (Amit et al., 2002Amit S. Hatzubai A. Birman Y. Andersen J.S. Ben-Shushan E. Mann M. Ben-Neriah Y. Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.Genes Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (577) Google Scholar; Jiang and Struhl, 1998Jiang J. Struhl G. Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb.Nature. 1998; 391: 493-496Crossref PubMed Scopus (535) Google Scholar; Li et al., 2012Li V.S.W. Ng S.S. Boersema P.J. Low T.Y. Karthaus W.R. Gerlach J.P. Mohammed S. Heck A.J.R. Maurice M.M. Mahmoudi T. Clevers H. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex.Cell. 2012; 149: 1245-1256Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar; Liu et al., 2002Liu C. Li Y. Semenov M. Han C. Baeg G.-H. Tan Y. Zhang Z. Lin X. He X. 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The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro.Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (795) Google Scholar; Wu et al., 2003Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Structure of a β-TrCP1-Skp1-β-catenin complex: destruction motif binding and lysine specificity of the SCF(β-TrCP1) ubiquitin ligase.Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Beta-catenin poly-ubiquitylation triggers its proteasomal degradation (Aberle et al., 1997Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. Beta-catenin is a target for the ubiquitin-proteasome pathway.EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2111) Google Scholar). The central DC scaffold, AXIN1, directly binds and assembles all components of the core DC, strongly boosting β-catenin phosphorylation (Dajani et al., 2003Dajani R. Fraser E. Roe S.M. Yeo M. Good V.M. Thompson V. Dale T.C. Pearl L.H. Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex.EMBO J. 2003; 22: 494-501Crossref PubMed Scopus (246) Google Scholar; Hamada et al., 1999Hamada F. Tomoyasu Y. Takatsu Y. Nakamura M. Nagai S. Suzuki A. Fujita F. Shibuya H. Toyoshima K. Ueno N. Akiyama T. Negative regulation of Wingless signaling by D-axin, a Drosophila homolog of axin.Science. 1999; 283: 1739-1742Crossref PubMed Scopus (167) Google Scholar; Ikeda et al., 1998Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin.EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1082) Google Scholar; Willert et al., 1999Willert K. Logan C.Y. Arora A. Fish M. Nusse R. A Drosophila Axin homolog, Daxin, inhibits Wnt signaling.Development. 1999; 126: 4165-4173Crossref PubMed Google Scholar), in part by suppressing phosphorylation of substrates competing with β-catenin (Gavagan et al., 2020Gavagan M. Fagnan E. Speltz E.B. Zalatan J.G. The scaffold protein Axin promotes signaling specificity within the Wnt pathway by suppressing competing kinase reactions.Cell Syst. 2020; 10: 515-525.e5Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Its low average cellular concentration (Lee et al., 2003Lee E. Salic A. Krüger R. Heinrich R. Kirschner M.W. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway.PLoS Biol. 2003; 1: E10Crossref PubMed Scopus (515) Google Scholar; Tan et al., 2012Tan C.W. Gardiner B.S. Hirokawa Y. Layton M.J. Smith D.W. Burgess A.W. Wnt signalling pathway parameters for mammalian cells.PLoS ONE. 2012; 7: e31882Crossref PubMed Scopus (89) Google Scholar) requires AXIN1 to form filamentous polymers for efficient β-catenin degradation (Fiedler et al., 2011Fiedler M. Mendoza-Topaz C. Rutherford T.J. Mieszczanek J. Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.Proc. Natl. Acad. Sci. U S A. 2011; 108: 1937-1942Crossref PubMed Scopus (138) Google Scholar). The second DC scaffold, APC, harbors up to ten β-catenin binding sites, a subset of which are phospho-regulated by the DC kinases (Eklof Spink et al., 2001Eklof Spink K. Fridman S.G. Weis W.I. Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-beta-catenin complex.EMBO J. 2001; 20: 6203-6212Crossref PubMed Scopus (109) Google Scholar; Ha et al., 2004Ha N.-C. Tonozuka T. Stamos J.L. Choi H.-J. Weis W.I. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.Mol. Cell. 2004; 15: 511-521Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar; Liu et al., 2006Liu J. Xing Y. Hinds T.R. Zheng J. Xu W. The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin.J. Mol. Biol. 2006; 360: 133-144Crossref PubMed Scopus (62) Google Scholar; Rubinfeld et al., 1993Rubinfeld B. Souza B. Albert I. Müller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Association of the APC gene product with beta-catenin.Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1165) Google Scholar; Su et al., 1993Su L.K. Vogelstein B. Kinzler K.W. Association of the APC tumor suppressor protein with catenins.Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1104) Google Scholar), as well as three AXIN1 docking motifs (Behrens et al., 1998Behrens J. Jerchow B.A. Würtele M. Grimm J. Asbrand C. Wirtz R. Kühl M. Wedlich D. Birchmeier W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.Science. 1998; 280: 596-599Crossref PubMed Scopus (1093) Google Scholar; Kishida et al., 1998Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin.J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar; Spink et al., 2000Spink K.E. Polakis P. Weis W.I. Structural basis of the Axin-adenomatous polyposis coli interaction.EMBO J. 2000; 19: 2270-2279Crossref PubMed Scopus (160) Google Scholar; Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref PubMed Scopus (621) Google Scholar; van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar; Figure S1A). Loss of varying numbers of these motifs by APC truncating mutations within the intestinal stem cell compartment confers high basal β-catenin levels and initiates up to 80% of colorectal cancers (CRCs) (Barker and Clevers, 2006Barker N. Clevers H. Mining the Wnt pathway for cancer therapeutics.Nat. Rev. Drug Discov. 2006; 5: 997-1014Crossref PubMed Scopus (637) Google Scholar; Kohler et al., 2008Kohler E.M. Derungs A. Daum G. Behrens J. 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Loss of beta-catenin regulation by the APC tumor suppressor protein correlates with loss of structure due to common somatic mutations of the gene.Cancer Res. 1997; 57: 4624-4630PubMed Google Scholar; Zhang and Shay, 2017Zhang L. Shay J.W. Multiple roles of APC and its therapeutic implications in colorectal cancer.J. Natl. Cancer Inst. 2017; 109: 55Crossref Scopus (144) Google Scholar), the third most common cancer type worldwide (Bray et al., 2018Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2018; 68: 394-424Crossref PubMed Scopus (43044) Google Scholar). Although the function of AXIN1 in the DC is relatively well understood, that of APC is less clear (van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar). Several non-mutually exclusive functions have been proposed for APC, among them the capture of β-catenin in the cytoplasm for its modification (Ha et al., 2004Ha N.-C. Tonozuka T. Stamos J.L. Choi H.-J. Weis W.I. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.Mol. Cell. 2004; 15: 511-521Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) or cytoplasmic retention (Krieghoff et al., 2006Krieghoff E. Behrens J. Mayr B. Nucleo-cytoplasmic distribution of β-catenin is regulated by retention.J. Cell Sci. 2006; 119: 1453-1463Crossref PubMed Scopus (209) Google Scholar; Roberts et al., 2011Roberts D.M. Pronobis M.I. Poulton J.S. Waldmann J.D. Stephenson E.M. Hanna S. Peifer M. Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.Mol. Biol. Cell. 2011; 22: 1845-1863Crossref PubMed Scopus (75) Google Scholar), the transfer of phosphorylated β-catenin from AXIN1 to the ubiquitin-proteasome system (Kimelman and Xu, 2006Kimelman D. Xu W. Beta-catenin destruction complex: insights and questions from a structural perspective.Oncogene. 2006; 25: 7482-7491Crossref PubMed Scopus (469) Google Scholar), or the protection of β-catenin from de-phosphorylation (Su et al., 2008Su Y. Fu C. Ishikawa S. Stella A. Kojima M. Shitoh K. Schreiber E.M. Day B.W. Liu B. APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase.Mol. Cell. 2008; 32: 652-661Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) or de-ubiquitylation (Novellasdemunt et al., 2017Novellasdemunt L. Foglizzo V. Cuadrado L. Antas P. Kucharska A. Encheva V. Snijders A.P. Li V.S.W. USP7 is a tumor-specific WNT activator for APC-mutated colorectal cancer by mediating β-catenin deubiquitination.Cell Rep. 2017; 21: 612-627Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Our understanding of the DC is shaped largely by genetic, cell biological, cellular biochemical, and structural studies (Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref PubMed Scopus (621) Google Scholar; van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar; and references above). A minimal DC has been constructed in mammalian cells (Pronobis et al., 2017Pronobis M.I. Deuitch N. Posham V. Mimori-Kiyosue Y. Peifer M. Reconstituting regulation of the canonical Wnt pathway by engineering a minimal β-catenin destruction machine.Mol. Biol. Cell. 2017; 28: 41-53Crossref PubMed Google Scholar), and the reconstitution of DC function in mammalian cell (Su et al., 2008Su Y. Fu C. Ishikawa S. Stella A. Kojima M. Shitoh K. Schreiber E.M. Day B.W. Liu B. APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase.Mol. Cell. 2008; 32: 652-661Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) or Xenopus egg (Salic et al., 2000Salic A. Lee E. Mayer L. Kirschner M.W. Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts.Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) extracts has been the closest step toward achieving full DC reconstitution in vitro. Still, both these systems offer limited control over the factors present in the experimental setup. The biochemical reconstitution of the DC from purified components, thus far hampered by the lack of purified, recombinant, full-length human AXIN1 and APC, has long been sought after, as it would enable the controlled interrogation of DC function in a reductionist system without confounding cellular factors. Here we describe the in vitro reconstitution of the core DC and recapitulate β-catenin capture, phosphorylation, ubiquitylation, and degradation, using purified components. We demonstrate that oncogenic APC mutations in CRC impart deficiencies to DC assembly and activities. To interrogate the mechanism of the core DC, we expressed the recombinant human complex in insect cells and affinity-purified it through a C-terminal double-StrepII tag on AXIN1. We included AXIN1 either in its wild-type or non-polymerizable mutant form (M3) (Fiedler et al., 2011Fiedler M. Mendoza-Topaz C. Rutherford T.J. Mieszczanek J. Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.Proc. Natl. Acad. Sci. U S A. 2011; 108: 1937-1942Crossref PubMed Scopus (138) Google Scholar). All full-length DC components (AXIN1, APC, CK1α, GSK3β, and β-catenin) were present in the purified complex (Figure 1B). However, loss of AXIN1 polymerization significantly reduced β-catenin recruitment (Figures 1B, 1C, and S1B), in agreement with the essential role of AXIN1 self-assembly in destabilizing β-catenin in vivo (Fiedler et al., 2011Fiedler M. Mendoza-Topaz C. Rutherford T.J. Mieszczanek J. Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.Proc. Natl. Acad. Sci. U S A. 2011; 108: 1937-1942Crossref PubMed Scopus (138) Google Scholar). Phosphorylation of β-catenin, APC, and AXIN1 is critical to the DC function (Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref PubMed Scopus (621) Google Scholar; van Kappel and Maurice, 2017van Kappel E.C. Maurice M.M. Molecular regulation and pharmacological targeting of the β-catenin destruction complex.Br. J. Pharmacol. 2017; 174: 4575-4588Crossref PubMed Scopus (39) Google Scholar). Immunoblotting revealed that a subpopulation of β-catenin within the complex was phosphorylated on the authentic phosphodegron sites targeted by CK1α and GSK3β (S45 and S33/S37/T41, respectively; Figure S1B; Liu et al., 2002Liu C. Li Y. Semenov M. Han C. Baeg G.-H. Tan Y. Zhang Z. Lin X. He X. Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar). Loss of AXIN1 polymerization also significantly reduced degron phosphorylation (Figures 1C and S1B). Treatment of the purified DC with λ-phosphatase increased APC and AXIN1 mobility in SDS-PAGE and abolished detectable β-catenin phosphorylation (Figures S1C). Conversely, ATP addition reduced AXIN1 mobility and further augmented β-catenin phosphorylation, showing that phosphorylation of these components was not saturated (Figure S1C). We next analyzed the DC’s mass by size exclusion chromatography coupled to in-line multi-angle light scattering (SEC-MALS). AXIN1 (92 kDa) is expected to bind a single copy each of β-catenin (85 kDa), CK1α (39 kDa), and GSK3β (47 kDa). Disregarding AXIN1 and APC multimerization (Fiedler et al., 2011Fiedler M. Mendoza-Topaz C. Rutherford T.J. Mieszczanek J. Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.Proc. Natl. Acad. Sci. U S A. 2011; 108: 1937-1942Crossref PubMed Scopus (138) Google Scholar; Kunttas-Tatli et al., 2014Kunttas-Tatli E. Roberts D.M. McCartney B.M. Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.Mol. Biol. Cell. 2014; 25: 3424-3436Crossref PubMed Google Scholar), we reasoned that up to three AXIN1 molecules could be bound by a single APC molecule (312 kDa) through APC’s SAMP repeats. APC would bind up to ten β-catenin molecules through its 15Rs and 20Rs. The resulting overall theoretical molecular weight would amount to 1,951 kDa (Figure S1A). At its highest attainable concentration, the DC displayed an average molecular weight of 2,395 ± 141 kDa (Figures 1D and S1D–S1F), indicating additional higher order contributions to DC stoichiometry. Dilution of the complex decreased the measured molecular weights, in line with the anticipated concentration dependency of complex assembly (Figures 1D and S1D–S1F). The size of the DC was not saturated at its highest concentration, but we were unable to concentrate the complex by ultrafiltration. AXIN1 polymerization is a major contributor to the overall size of the DC: even at a higher concentration than that for the wild-type DC, the AXIN1 M3 mutant complex displayed an average molecular weight of only 1,072 ± 68 kDa, and its dilution had no impact on its overall mass (Figures 1D and S1D–S1F). As for affinity purification, the eluting complex showed an increased β-catenin occupancy with wild-type compared with M3 AXIN1 (Figures S1E and S1F). We next used mass photometry to measure the DC’s mass distribution at the single-molecule level (Sonn-Segev et al., 2020Sonn-Segev A. Belacic K. Bodrug T. Young G. VanderLinden R.T. Schulman B.A. Schimpf J. Friedrich T. Dip P.V. Schwartz T.U. et al.Quantifying the heterogeneity of macromolecular machines by mass photometry.Nat. Commun. 2020; 11: 1772Crossref PubMed Scopus (49) Google Scholar; Young et al., 2018Young G. Hundt N. Cole D. Fineberg A. Andrecka J. Tyler A. Olerinyova A. Ansari A. Marklund E.G. Collier M.P. et al.Quantitative mass imaging of single biological macromolecules.Science. 2018; 360: 423-427Crossref PubMed Scopus (193) Google Scholar). We first performed SEC-MALS on DCs containing either wild-type or M3 AXIN1 (Figure 1E), with results comparable with those shown in Figure 1D. We next analyzed the SEC-MALS input material and fractions across the elution peaks (Figure 1E, indicated by colored dots on gels) using mass photometry (Figure 1F). Molecular weight distributions were broad. The dominance of events below 0.2 MDa indicated DC dissociation upon dilution for mass photometry (<100 nM) (Sonn-Segev et al., 2020Sonn-Segev A. Belacic K. Bodrug T. Young G. VanderLinden R.T. Schulman B.A. Schimpf J. Friedrich T. Dip P.V. Schwartz T.U. et al.Quantifying the heterogeneity of macromolecular machines by mass photometry.Nat. Commun. 2020; 11: 1772Crossref PubMed Scopus (49) Google Scholar), even within the short time frame (≈5 min). This generated multiple lower molecular weight events from the parental complex, in line with the dynamic, concentration-dependent assembly of the DC (Barua and Hlavacek, 2013Barua D. Hlavacek W.S. Modeling the effect of APC truncation on destruction complex function in colorectal cancer cells.PLoS Comput. Biol. 2013; 9: e1003217Crossref PubMed Scopus (17) Google Scholar; Nong et al., 2021Nong J. Kang K. Shi Q. Zhu X. Tao Q. Chen Y.-G. Phase separation of Axin organizes the β-catenin destruction complex.J. Cell Biol. 2021; 220: e202012112Crossref PubMed Google Scholar; Pronobis et al., 2017Pronobis M.I. Deuitch N. Posham V. Mimori-Kiyosue Y. Peifer M. Reconstituting regulation of the canonical Wnt pathway by engineering a minimal β-catenin destruction machine.Mol. Biol. Cell. 2017; 28: 41-53Crossref PubMed Google Scholar; Schaefer and Peifer, 2019Schaefer K.N. Peifer M. Wnt/beta-catenin signaling regulation and a role for biomolecular condensates.Dev. Cell. 2019; 48: 429-444Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Nonetheless, we still detected high-molecular weight species (Figure 1F). Although most events measured for DC AXIN1 M3 were less than 1.5 MDa, the wild-type DC displayed a broader mass distribution ranging up to ≈3 MDa (Figure 1F). In conclusion, mass photometry further illustrates the importance of AXIN1 polymerization in DC assembly. We performed in silico modeling to predict possible stoichiometries within the DC that satisfy both the molecular weights observed in SEC-MALS and constraints on the basis of our own and published findings on DC assembly (Figure 1G; Table S1; see STAR Methods for details). Modeling suggests that the DC can attain different stoichiometries for a given molecular weight range. Although AXIN1 polymerization and the APC/AXIN1 ratio are important stoichiometry determinants, the degree of β-catenin incorporation contributes most to the stoichiometry variability (Figure 1G; Table S1). Taken together, we show that AXIN1 polymerization governs the concentration-dependent assembly of the DC, recruitment of β-catenin, and complex composition. We next assessed the contribution of APC to DC assembly by purifying different DC variants. The kinases robustly co-purified with AXIN1, regardless of APC’s presence (Figure 2B, lanes 17 and 13). Although only s" @default.
• W3192327720 created "2021-08-16" @default.
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• W3192327720 date "2021-08-01" @default.
• W3192327720 modified "2023-10-14" @default.
• W3192327720 title "Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation" @default.
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• W3192327720 doi "https://doi.org/10.1016/j.molcel.2021.07.013" @default.
• W3192327720 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8403986" @default.
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• W3192327720 hasPublicationYear "2021" @default.

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