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• W2064027874 abstract "Degradation of cytosolic β-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that β-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound β-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses β-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-β-catenin. Subsequently, newly synthesized β-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors. Degradation of cytosolic β-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that β-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound β-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses β-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-β-catenin. Subsequently, newly synthesized β-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors. The composition of the Axin complex does not change upon Wnt signaling GSK3 is not inhibited by Wnt; phospho β-catenin accumulates in Axin complex β-catenin is removed from Axin complex by the proteasome upon ubiquitination Axin complex remains compositionally intact in APC mutant colorectal cancer The canonical Wnt (Wnt/β-catenin) signaling pathway controls many biological processes, including cell fate determination, cell proliferation, and stem cell maintenance (Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar). Deregulation of this pathway occurs in cancer and underlies multiple hereditary syndromes (Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar, MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar). The key regulatory step involves the phosphorylation, ubiquitination, and subsequent degradation of its downstream effector protein, β-catenin, by a dedicated cytoplasmic destruction complex. This complex consists of the central scaffold protein Axin and three other core components, adenomatous polyposis coli (APC) and the kinases glycogen synthase kinase-3 alpha/beta (GSK-3) and casein kinase-1 (CKI). Mutations in components of the β-catenin destruction complex (APC, AXIN, or β-catenin) result in cancer (Kinzler and Vogelstein, 1996Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4269) Google Scholar, Korinek et al., 1997Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2927) Google Scholar, Liu et al., 2000Liu W. Dong X. Mai M. Seelan R.S. Taniguchi K. Krishnadath K.K. Halling K.C. Cunningham J.M. Boardman L.A. Qian C. et al.Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling.Nat. Genet. 2000; 26: 146-147Crossref PubMed Scopus (441) Google Scholar, Morin et al., 1997Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3496) Google Scholar, Rubinfeld et al., 1996Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly.Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1296) Google Scholar), most notably of the colon. In resting cells, despite the gene being continuously transcribed, vanishingly low levels of free β-catenin protein are present in the cytosol. This pool of β-catenin is efficiently captured by the destruction complex and phosphorylated by CKI at Ser45, which in turn primes GSK3 phosphorylation of β-catenin on the more N-terminal Thr41, Ser37, and Ser33 residues (Liu et al., 2002Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1660) Google Scholar). Phosphorylated β-catenin is ubiquitinated by the F-box-containing protein β-TrCP ubiquitin E3 ligase to be degraded by the proteasome (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 (2153) Google Scholar, Kitagawa et al., 1999Kitagawa M. Hatakeyama S. Shirane M. Matsumoto M. Ishida N. Hattori K. Nakamichi I. Kikuchi A. Nakayama K. Nakayama K. An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin.EMBO J. 1999; 18: 2401-2410Crossref PubMed Scopus (479) Google Scholar). Axin1 is the rate-limiting factor of the destruction complex (Lee et al., 2003Lee E. Salic A. Kruger 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 (531) Google Scholar). Axin1 directly interacts with all other core components of the destruction complex (β-catenin, APC, CKα, and GSK3), thus being the central scaffold of the complex (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 (1101) 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: 10823-10826Crossref PubMed Scopus (442) Google Scholar, Liu et al., 2002Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1660) Google Scholar, Sakanaka et al., 1998Sakanaka C. Weiss J.B. Williams L.T. Bridging of beta-catenin and glycogen synthase kinase-3beta by axin and inhibition of beta-catenin-mediated transcription.Proc. Natl. Acad. Sci. USA. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar). As the least abundant component, Axin1 can regulate its rapid assembly and disassembly. For this reason, it has been proposed that degradation of Axin1 in Wnt-activated cells may be the immediate cause of β-catenin stabilization (Mao et al., 2001Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, Tolwinski et al., 2003Tolwinski N.S. Wehrli M. Rives A. Erdeniz N. DiNardo S. Wieschaus E. Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.Dev. Cell. 2003; 4: 407-418Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Although multiple roles have been proposed for the genetically essential APC protein, there is no consensus as to its key activity. Wnt ligands bind to the frizzled (FZD) and low-density-lipoprotein-related protein 5/6 (LRP5/6) coreceptor complex to activate the canonical Wnt signaling pathway. Through an incompletely resolved mechanism that involves Dishevelled, the activated receptor complex disrupts or functionally inactivates the destruction complex, leading to the accumulation and nuclear translocation of β-catenin. In the nucleus, β-catenin engages TCF/LEF transcription factors to activate the Wnt transcriptional program (Molenaar et al., 1996Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar, Behrens et al., 1996Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Functional interaction of beta-catenin with the transcription factor LEF-1.Nature. 1996; 382: 638-642Crossref PubMed Scopus (2590) Google Scholar). Several models describe the events following Wnt receptor activation that lead to stabilization of β-catenin. Sequestration of Axin1 by binding LRP5/6 reduces the availability of cytoplasmic destruction complexes, thereby causing β-catenin accumulation. Membrane translocation of Axin1 may also mediate its Wnt-induced dephosphorylation and destabilization (Mao et al., 2001Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, Zeng et al., 2005Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.Nature. 2005; 438: 873-877Crossref PubMed Scopus (653) Google Scholar). Mediated by activated Wnt receptors or Dishevelled (Lee et al., 2003Lee E. Salic A. Kruger 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 (531) Google Scholar, Mao et al., 2001Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, Tolwinski et al., 2003Tolwinski N.S. Wehrli M. Rives A. Erdeniz N. DiNardo S. Wieschaus E. Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.Dev. Cell. 2003; 4: 407-418Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Also, the poly-ADP-ribosylating enzyme tankyrase can mediate Axin1 degradation (Huang et al., 2009Huang S.M. Mishina Y.M. Liu S. Cheung A. Stegmeier F. Michaud G.A. Charlat O. Wiellette E. Zhang Y. Wiessner S. et al.Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.Nature. 2009; 461: 614-620Crossref PubMed Scopus (1532) Google Scholar). By contrast, SUMOylation of Axin1 was reported to protect it from polyubiquitination and increase its stability (Kim et al., 2008Kim M.J. Chia I.V. Costantini F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability.FASEB J. 2008; 22: 3785-3794Crossref PubMed Scopus (37) Google Scholar). An endocytic adaptor protein Dab2 prevents Axin1 membrane translocation, thereby leading to Axin1 stabilization (Jiang et al., 2009Jiang Y. Luo W. Howe P.H. Dab2 stabilizes Axin and attenuates Wnt/beta-catenin signaling by preventing protein phosphatase 1 (PP1)-Axin interactions.Oncogene. 2009; 28: 2999-3007Crossref PubMed Scopus (39) Google Scholar). Dishevelled, which binds Axin1 directly, may disrupt the destruction complex upon Wnt activation (Liu et al., 2005Liu X. Rubin J.S. Kimmel A.R. Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins.Curr. Biol. 2005; 15: 1989-1997Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, Logan and Nusse, 2004Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4221) Google Scholar, Malbon and Wang, 2006Malbon C.C. Wang H.Y. Dishevelled: a mobile scaffold catalyzing development.Curr. Top. Dev. Biol. 2006; 72: 153-166Crossref PubMed Scopus (99) Google Scholar). Alternatively, Frat/GBP family members may compete with Axin1 for GSK3 binding, thus disrupting the destruction complex (van Amerongen and Berns, 2005van Amerongen R. Berns A. Re-evaluating the role of Frat in Wnt-signal transduction.Cell Cycle. 2005; 4: 1065-1072PubMed Google Scholar, van Amerongen et al., 2005van Amerongen R. Nawijn M. Franca-Koh J. Zevenhoven J. van der Gulden H. Jonkers J. Berns A. Frat is dispensable for canonical Wnt signaling in mammals.Genes Dev. 2005; 19: 425-430Crossref PubMed Scopus (64) Google Scholar). CKI and GSK3 can phosphorylate Axin1 and APC, enhancing their binding affinity for GSK3 and β-catenin. Wnt stimulation results in dephosphorylation of both Axin1 and APC. The catalytic subunits of the phosphatases PP1 and PP2A directly bind and dephosphorylate Axin1, promoting the disassembly of the destruction complex (Luo et al., 2007Luo W. Peterson A. Garcia B.A. Coombs G. Kofahl B. Heinrich R. Shabanowitz J. Hunt D.F. Yost H.J. Virshup D.M. Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex.EMBO J. 2007; 26: 1511-1521Crossref PubMed Scopus (97) Google Scholar, Strovel et al., 2000Strovel E.T. Wu D. Sussman D.J. Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription.J. Biol. Chem. 2000; 275: 2399-2403Crossref PubMed Scopus (93) Google Scholar). In vitro phosphorylation of β-catenin by GSK3 is inhibited by PPPSPxS motif peptides or by phosphorylated LRP6 cytoplasmic domain (Cselenyi et al., 2008Cselenyi C.S. Jernigan K.K. Tahinci E. Thorne C.A. Lee L.A. Lee E. LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of beta-catenin.Proc. Natl. Acad. Sci. USA. 2008; 105: 8032-8037Crossref PubMed Scopus (162) Google Scholar, Piao et al., 2008Piao S. Lee S.H. Kim H. Yum S. Stamos J.L. Xu Y. Lee S.J. Lee J. Oh S. Han J.K. et al.Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-catenin signaling.PLoS ONE. 2008; 3: e4046Crossref PubMed Scopus (161) Google Scholar, Wu et al., 2009Wu G. Huang H. Garcia Abreu J. He X. Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.PLoS ONE. 2009; 4: e4926Crossref PubMed Scopus (163) Google Scholar). In addition, suppression of GSK3 kinase activity by protein kinase B (PKB) directly or through Dishevelled may mediate Wnt signal transduction (Desbois-Mouthon et al., 2001Desbois-Mouthon C. Cadoret A. Blivet-Van Eggelpoël M.J. Bertrand F. Cherqui G. Perret C. Capeau J. Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation.Oncogene. 2001; 20: 252-259Crossref PubMed Scopus (267) Google Scholar, Fukumoto et al., 2001Fukumoto S. Hsieh C.M. Maemura K. Layne M.D. Yet S.F. Lee K.H. Matsui T. Rosenzweig A. Taylor W.G. Rubin J.S. et al.Akt participation in the Wnt signaling pathway through Dishevelled.J. Biol. Chem. 2001; 276: 17479-17483Crossref PubMed Scopus (311) Google Scholar). CKIα/GSK3-phosphorylated β-catenin can be dephosphorylated by phosphatase PP2A upon Wnt induction (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 (135) Google Scholar). Taelman et al. propose that sequestration of GSK3 from the cytosol into multivesicular bodies inhibits GSK3 activity during Wnt signal transduction (Taelman et al., 2010Taelman V.F. Dobrowolski R. Plouhinec J.L. Fuentealba L.C. Vorwald P.P. Gumper I. Sabatini D.D. De Robertis E.M. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.Cell. 2010; 143: 1136-1148Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). Roberts et al. suggest that disassembly of the destruction complex by APC through transferring phosphorylated β-catenin to the E3 ligase recycles the destruction complex for renewed β-catenin phosphorylation and degradation (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 (78) Google Scholar). Thus, all previously proposed models (MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar) either assume a physical dissociation of the β-catenin destruction complex or propose an interference with β-catenin phosphorylation, leading to stabilization of β-catenin. All studies cited above, however, utilize overexpression strategies. Studies on the endogenous complex are hampered by the fact that Axin1 is expressed at vanishingly low levels, whereas APC, CK1, and GSK3, as well as β-catenin, occur abundantly outside of the destruction complex. Here, we study the endogenous destruction complex during Wnt signaling. HEK293T cells carry an intact Wnt signaling cascade. We previously generated high-affinity immunoprecipitating (IP)/western blotting monoclonal antibodies against Axin1 (Ng et al., 2009Ng S.S. Mahmoudi T. Danenberg E. Bejaoui I. de Lau W. Korswagen H.C. Schutte M. Clevers H. Phosphatidylinositol 3-kinase signaling does not activate the wnt cascade.J. Biol. Chem. 2009; 284: 35308-35313Crossref PubMed Scopus (120) Google Scholar). Pooled HEK293T cells from two 15 cm culture dishes allow visualization of endogenous Axin1 in a single lane by western blotting after immunoprecipitation (Ng et al., 2009Ng S.S. Mahmoudi T. Danenberg E. Bejaoui I. de Lau W. Korswagen H.C. Schutte M. Clevers H. Phosphatidylinositol 3-kinase signaling does not activate the wnt cascade.J. Biol. Chem. 2009; 284: 35308-35313Crossref PubMed Scopus (120) Google Scholar). This strategy allowed us to probe the composition of the destruction complex in Wnt-active versus nonactive HEK293T cells. Axin1 protein is degraded upon Wnt stimulation (Jiang et al., 2009Jiang Y. Luo W. Howe P.H. Dab2 stabilizes Axin and attenuates Wnt/beta-catenin signaling by preventing protein phosphatase 1 (PP1)-Axin interactions.Oncogene. 2009; 28: 2999-3007Crossref PubMed Scopus (39) Google Scholar, Kim et al., 2008Kim M.J. Chia I.V. Costantini F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability.FASEB J. 2008; 22: 3785-3794Crossref PubMed Scopus (37) Google Scholar, Mao et al., 2001Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). We stimulated HEK293T cells with Wnt3A-conditioned medium or control medium and monitored Axin1 protein and cytosolic β-catenin by western blot analysis (Figure S1A available online and Figure 1A ). Of note, the overwhelming amount of β-catenin resides in the membrane-bound E-cadherin complex, a highly stable pool that is irrelevant to Wnt signaling (van de Wetering et al., 2001van de Wetering M. Barker N. Harkes I.C. van der Heyden M. Dijk N.J. Hollestelle A. Klijn J.G. Clevers H. Schutte M. Mutant E-cadherin breast cancer cells do not display constitutive Wnt signaling.Cancer Res. 2001; 61: 278-284PubMed Google Scholar). To increase detection sensitivity, we performed Axin1 immunoprecipitation on whole-cell lysates prior to western blot analysis (Figures 1B and 1C). Though cytosolic accumulation of β-catenin was detectable as early as 30 min following Wnt treatment (Figure 1A), we first observed a significant decrease in endogenous Axin1 protein at 4 hr post-Wnt stimulation (Figures 1B, 1C, and S1A), implying that degradation of Axin1 protein is not causal to the initial activation of the Wnt pathway.Figure 1Wnt-Induced Stabilization of β-Catenin Occurs prior to and Independently of Axin1 DegradationShow full captionHEK293T cells were stimulated with Wnt according to the indicated time points.(A) Cytosolic β-catenin protein levels begin to accumulate 30 min after Wnt stimulation, peaking at ∼2 hr. GAPDH protein levels were used as loading control. Note that there is vanishingly detectable free β-catenin in cytosol in the absence of Wnt.(B) Axin1 immunoprecipitation was performed from whole-cell lysates for detection of endogenous Axin1 level upon Wnt induction. GAPDH input was used as input loading control.(C) Quantitation of Axin1 protein level relative to GAPDH. Axin1 degradation becomes significant at 4 hr post-Wnt induction. Error bars represent ±SD.(D) Wnt-induced activation of target genes CCND1, TCF7, ZCCHC12, EPHB3, and AXIN2 and, as control, ACTB was examined in HEK293T cells by quantitative RT-PCR at the indicated time points. Time course expression data are presented as fold induction normalized to GAPDH control in triplicate and are representative of at least two independent experiments. Error bars represent ±SD.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HEK293T cells were stimulated with Wnt according to the indicated time points. (A) Cytosolic β-catenin protein levels begin to accumulate 30 min after Wnt stimulation, peaking at ∼2 hr. GAPDH protein levels were used as loading control. Note that there is vanishingly detectable free β-catenin in cytosol in the absence of Wnt. (B) Axin1 immunoprecipitation was performed from whole-cell lysates for detection of endogenous Axin1 level upon Wnt induction. GAPDH input was used as input loading control. (C) Quantitation of Axin1 protein level relative to GAPDH. Axin1 degradation becomes significant at 4 hr post-Wnt induction. Error bars represent ±SD. (D) Wnt-induced activation of target genes CCND1, TCF7, ZCCHC12, EPHB3, and AXIN2 and, as control, ACTB was examined in HEK293T cells by quantitative RT-PCR at the indicated time points. Time course expression data are presented as fold induction normalized to GAPDH control in triplicate and are representative of at least two independent experiments. Error bars represent ±SD. See also Figure S1. To extend these findings, we examined the endogenous Wnt target genes AXIN2 (Lustig et al., 2002Lustig B. Jerchow B. Sachs M. Weiler S. Pietsch T. Karsten U. van de Wetering M. Clevers H. Schlag P.M. Birchmeier W. Behrens J. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors.Mol. Cell. Biol. 2002; 22: 1184-1193Crossref PubMed Scopus (838) Google Scholar), CCND1 (Tetsu and McCormick, 1999Tetsu O. McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells.Nature. 1999; 398: 422-426Crossref PubMed Scopus (3253) Google Scholar), EPHB3 (van de Wetering, M et al., 2002), TCF7 (Roose et al., 1999Roose J. Huls G. van Beest M. Moerer P. van der Horn K. Goldschmeding R. Logtenberg T. Clevers H. Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1.Science. 1999; 285: 1923-1926Crossref PubMed Scopus (402) Google Scholar), and ZCCHC12 (Mahmoudi et al., 2009Mahmoudi T. Li V.S. Ng S.S. Taouatas N. Vries R.G. Mohammed S. Heck A.J. Clevers H. The kinase TNIK is an essential activator of Wnt target genes.EMBO J. 2009; 28: 3329-3340Crossref PubMed Scopus (135) Google Scholar) by quantitative RT-PCR in a time course experiment (Figure 1D). Significant mRNA increases were detected from 0.5–2 hr post-Wnt stimulation (Figure 1D), when Axin1 protein levels were still unchanged. We then asked how the interaction between Axin1 and other components of the Wnt cascade is influenced by Wnt stimulation. We immunoprecipitated the Axin1 complex from HEK293T cells before and during Wnt treatment, followed by western blotting (Figure 2A ). In the absence of Wnt, Axin1 interacted with GSK3 (last panel) and APC (second panel), but not LRP6 (fourth panel). Phosphorylated LRP6 was only detected in the Axin1 complex after Wnt stimulation (third panel) (Mao et al., 2001Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, Tamai et al., 2004Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. A mechanism for Wnt coreceptor activation.Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, Tolwinski et al., 2003Tolwinski N.S. Wehrli M. Rives A. Erdeniz N. DiNardo S. Wieschaus E. Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.Dev. Cell. 2003; 4: 407-418Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Dishevelled 3 coimmunoprecipitated with Axin1 in the absence and presence of Wnt (fifth panel). In contrast to previous reports (Liu et al., 2005Liu X. Rubin J.S. Kimmel A.R. Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins.Curr. Biol. 2005; 15: 1989-1997Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, Logan and Nusse, 2004Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4221) Google Scholar), we did not find a significant decrease in binding of either APC (second panel) or GSK3β (last panel) to Axin1 in response to Wnt stimulation. This observation was inconsistent with models in which dissociation of the destruction complex or modulation of Axin1 binding to GSK3β or APC mediate functional inactivation of the destruction complex. β-catenin coimmunoprecipitating with Axin1 was hardly detectable in the absence of Wnt stimulation, highlighting the dynamic nature of the β-catenin/Axin1 interaction (Figure 2, eighth panel). Surprisingly, we found a significant Wnt-induced increase in β-catenin immunoprecipitating with Axin1. We further analyzed this β-catenin pool using antibodies that specifically recognize P-Ser45 β-catenin or P-Ser33/Ser37/Thr41 β-catenin (Figure 2, sixth and seventh panel). Counter to prediction, we found an increase in phosphorylated β-catenin in the Wnt-stimulated Axin1 complex (Figure S2A ). This finding was consistent with a previous report noting phosphorylated β-catenin in high-molecular-weight complexes, whereas non-phospho-β-catenin accumulated in a monomeric form upon Wnt signaling (Maher et al., 2010Maher M.T. Mo R. Flozak A.S. Peled O.N. Gottardi C.J. Beta-catenin phosphorylated at serine 45 is spatially uncoupled from beta-catenin phosphorylated in the GSK3 domain: implications for signaling.PLoS ONE. 2010; 5: e10184Crossref PubMed Scopus (74) Google Scholar). Thus, the critical kinases CK1 and GSK3β remain present and active within the destruction complex upon Wnt signaling. We also combined Axin1 immunoprecipitation with mass spectrometry (MS) to obtain a global picture of the Axin1 complex in HEK293T. Consistent with our IP results, we readily detected the core components of the destruction complex (APC, GSK3β, CK1, and β-catenin) in both Wnt-inactive and -activated cells (Table S1). Quantitative MS using a label-free approach with extracted ion chromatograms further confirmed a significant increase of β-catenin detected within the Axin complex after Wnt stimulation. Other components (e.g., APC, GSK3β, and CK1) remained unchanged (Figures S2C, S2D, and Table S2). Similar observations were reported recently (Hilger and Mann, 2012Hilger M. Mann M. Triple SILAC to determine stimulus specific interactions in the Wnt pathway.J. Proteome Res. 2012; 11: 982-994Crossref PubMed Scopus" @default.
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