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• W1987486570 abstract "Post-translational stabilization of β-catenin is a key step in Wnt signaling, but the features of β-catenin required for stabilization are incompletely understood. We show that forms of β-catenin lacking the unstructured C-terminal domain (CTD) show faster turnover than full-length or minimally truncated β-catenins. Mutants that exhibit faster turnover show enhanced association with axin in co-transfected cells, and excess CTD polypeptide can compete binding of the β-catenin armadillo (arm) repeat domain to axin in vitro, indicating that the CTD may restrict β-catenin binding to the axin-scaffold complex. Fluorescent resonance energy transmission (FRET) analysis of cyan fluorescent protein (CFP)-arm-CTD-yellow fluorescent protein β-catenin reveals that the CTD of β-catenin can become spatially close to the N-terminal arm repeat region of β-catenin. FRET activity is strongly diminished by the coexpression of β-catenin binding partners, indicating that an unliganded groove is absolutely required for an orientation that allows FRET. Amino acids 733–759 are critical for β-catenin FRET activity and stability. These data indicate that an N-terminal orientation of the CTD is required for β-catenin stabilization and suggest a model where the CTD extends toward the N-terminal arm repeats, shielding these repeats from the β-catenin destruction complex. Post-translational stabilization of β-catenin is a key step in Wnt signaling, but the features of β-catenin required for stabilization are incompletely understood. We show that forms of β-catenin lacking the unstructured C-terminal domain (CTD) show faster turnover than full-length or minimally truncated β-catenins. Mutants that exhibit faster turnover show enhanced association with axin in co-transfected cells, and excess CTD polypeptide can compete binding of the β-catenin armadillo (arm) repeat domain to axin in vitro, indicating that the CTD may restrict β-catenin binding to the axin-scaffold complex. Fluorescent resonance energy transmission (FRET) analysis of cyan fluorescent protein (CFP)-arm-CTD-yellow fluorescent protein β-catenin reveals that the CTD of β-catenin can become spatially close to the N-terminal arm repeat region of β-catenin. FRET activity is strongly diminished by the coexpression of β-catenin binding partners, indicating that an unliganded groove is absolutely required for an orientation that allows FRET. Amino acids 733–759 are critical for β-catenin FRET activity and stability. These data indicate that an N-terminal orientation of the CTD is required for β-catenin stabilization and suggest a model where the CTD extends toward the N-terminal arm repeats, shielding these repeats from the β-catenin destruction complex. The protein β-catenin serves two fundamental roles in the formation and maintenance of tissues. At the cell surface, β-catenin binds the cytoplasmic domain of cadherin-type adhesion receptors, allowing cells to engage their neighbors through robust intercellular adhering junctions. In the cytoplasm and nucleus, a cadherin-independent pool of β-catenin interacts with TCF 3The abbreviations used are: TCFT cell factorGSKglycogen synthase kinaseAPCadenomatous polyposis coliFRETfluorescent resonance energy transmissionarmarmadillo repeat domainGSTglutathione S-transferaseICATinhibitor of catenin and TCFCTDC-terminal domainCBDcatenin binding domainCFPcyan fluorescent proteinYFPyellow fluorescent proteinGAPDHglyceraldehyde-3-phosphate dehydrogenase. -type transcription factors to activate genes that produce cells with distinct identities. The abundance of this cytosolic/nuclear pool of β-catenin is largely determined by the presence of extracellular Wnt factors, which initiate a signaling cascade that prevents the continual destruction of cadherin-free β-catenin. T cell factor glycogen synthase kinase adenomatous polyposis coli fluorescent resonance energy transmission armadillo repeat domain glutathione S-transferase inhibitor of catenin and TCF C-terminal domain catenin binding domain cyan fluorescent protein yellow fluorescent protein glyceraldehyde-3-phosphate dehydrogenase. The destruction of β-catenin is carried out by a highly coordinated series of phosphorylation events. In the absence of Wnt, the N terminus of β-catenin is sequentially phosphorylated by casein kinase 1α and glycogen synthase kinase 3β (GSK3β) (1Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1687) Google Scholar, 2Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1020) Google Scholar). Phosphorylation of β-catenin by GSK at residues serine 33 and 37 allows recognition by the E3-ligase component βTrCP, which when part of the SCFβTrCP complex, catalyzes the ubiquitylation and rapid degradation of β-catenin (3Hart M. Concordet J.P. Lassot I. Albert I. del los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Benarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). This phosphorylation-dependent degradation of β-catenin depends on the scaffold protein, axin, and the tumor suppressor APC. Axin has binding sites for casein kinase 1α, GSK3β, β-catenin, and APC, so that phosphorylation of axin by casein kinase 1α and GSK3β increases binding to β-catenin (4Jho E. Lomvardas S. Costantini F. Biochem. Biophys. Res. Commun. 1999; 266: 28-35Crossref PubMed Scopus (75) Google Scholar, 5Willert K. Shibamoto S. Nusse R. Genes Dev. 1999; 13: 1768-1773Crossref PubMed Scopus (300) Google Scholar, 6Luo W. Peterson A. Garcia B.A. Coombs G. Kofahl B. Heinrich R. Shabanowitz J. Hunt D.F. Yost H.J. Virshup D.M. EMBO J. 2007; 26: 1511-1521Crossref PubMed Scopus (98) Google Scholar), allowing the N-terminal region of β-catenin to be a more efficient substrate of casein kinase 1α and GSK3β (7Dajani R. Fraser E. Roe S.M. Yeo M. Good V.M. Thompson V. Dale T.C. Pearl L.H. EMBO J. 2003; 22: 494-501Crossref PubMed Scopus (260) Google Scholar). Axin also promotes phosphorylation of APC by casein kinase 1ϵ and GSK3β (8Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1310) Google Scholar, 9Rubinfeld B. Tice D.A. Polakis P. J. Biol. Chem. 2001; 276: 39037-39045Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), increasing the affinity of APC for β-catenin (10Xing Y. Clements W.K. Kimelman D. Xu W. Genes Dev. 2003; 17: 2753-2764Crossref PubMed Scopus (198) Google Scholar, 11Ha N.C. Tonozuka T. Stamos J.L. Choi H.J. Weis W.I. Mol. Cell. 2004; 15: 511-521Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 12Xing Y. Clements W.K. Le Trong I. Hinds T.R. Stenkamp R. Kimelman D. Xu W. Mol. Cell. 2004; 15: 523-533Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Because phospho-APC binds to β-catenin with higher affinity than phospho-axin, it is thought that ordered phosphorylations within the axin-APC complex may control the flow of β-catenin through this complex (10Xing Y. Clements W.K. Kimelman D. Xu W. Genes Dev. 2003; 17: 2753-2764Crossref PubMed Scopus (198) Google Scholar, 11Ha N.C. Tonozuka T. Stamos J.L. Choi H.J. Weis W.I. Mol. Cell. 2004; 15: 511-521Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Once β-catenin is bound to APC, APC ensures targeting of N-terminal-phosphorylated β-catenin (Ser(P)-33 and Ser(P)-37) to the proteasome by protecting this βTrCP recognition epitope from the phosphatase PP2A (13Su Y. Fu C. Ishikawa S. Stella A. Kojima M. Shitoh K. Schreiber E.M. Day B.W. Liu B. Mol. Cell. 2008; 32: 652-661Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Understanding how β-catenin moves through the axin-APC-scaffold destruction complex requires an appreciation of β-catenin structure. β-Catenin belongs to the armadillo family of proteins, which are characterized by a central domain consisting of a repeating 42-amino acid motif, termed the “arm repeat” (14Riggleman B. Wieschaus E. Schedl P. Genes Dev. 1989; 3: 96-113Crossref PubMed Scopus (235) Google Scholar). X-ray crystallographic analysis of the β-catenin central armadillo domain shows that its 12 arm repeats form a superhelix of helices that create a long positively charged groove (15Huber A.H. Nelson W.J. Weis W.I. Cell. 1997; 90: 871-882Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Interestingly, these positive charges are critical for β-catenin binding to many of its negatively charged unstructured ligands such as the cadherin adhesion receptor, axin and APC degradation machinery components, or TCF-DNA binding factors (10Xing Y. Clements W.K. Kimelman D. Xu W. Genes Dev. 2003; 17: 2753-2764Crossref PubMed Scopus (198) Google Scholar, 12Xing Y. Clements W.K. Le Trong I. Hinds T.R. Stenkamp R. Kimelman D. Xu W. Mol. Cell. 2004; 15: 523-533Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 16von Kries J.P. Winbeck G. Asbrand C. Schwarz-Romond T. Sochnikova N. Dell'Oro A. Behrens J. Birchmeier W. Nat. Struct. Biol. 2000; 7: 800-807Crossref PubMed Scopus (169) Google Scholar, 17Graham T.A. Weaver C. Mao F. Kimelman D. Xu W. Cell. 2000; 103: 885-896Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 18Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Phosphorylation of these binding partners introduces additional negative charges that enhance interactions with the positively charged groove of β-catenin, thereby increasing binding affinity (10Xing Y. Clements W.K. Kimelman D. Xu W. Genes Dev. 2003; 17: 2753-2764Crossref PubMed Scopus (198) Google Scholar, 11Ha N.C. Tonozuka T. Stamos J.L. Choi H.J. Weis W.I. Mol. Cell. 2004; 15: 511-521Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 12Xing Y. Clements W.K. Le Trong I. Hinds T.R. Stenkamp R. Kimelman D. Xu W. Mol. Cell. 2004; 15: 523-533Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 19Choi H.J. Huber A.H. Weis W.I. J. Biol. Chem. 2006; 281: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Thus, the extent to which β-catenin is used in adhesive or nuclear signaling complexes will critically depend on the phosphorylation state of these major β-catenin ligands (for review, see Ref. 20Daugherty R.L. Gottardi C.J. Physiology. 2007; 22: 303-309Crossref PubMed Scopus (208) Google Scholar). During Wnt activation, GSK3β activity is locally inhibited within the axin complex (21Cselenyi C.S. Jernigan K.K. Tahinci E. Thorne C.A. Lee L.A. Lee E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8032-8037Crossref PubMed Scopus (164) Google Scholar, 22Piao S. Lee S.H. Kim H. Yum S. Stamos J.L. Xu Y. Lee S.J. Lee J. Oh S. Han J.K. Park B.J. Weis W.I. Ha N.C. PLoS ONE. 2008; 3: e4046Crossref PubMed Scopus (165) Google Scholar, 23Wu G. Huang H. Garcia Abreu J. He X. PLoS ONE. 2009; 4: e4926Crossref PubMed Scopus (167) Google Scholar), allowing β-catenin to escape this phosphorylation-dependent degradation mechanism and accumulate in both cytoplasmic and nuclear compartments (24Peifer M. Sweeton D. Casey M. Wieschaus E. Development. 1994; 120: 369-380Crossref PubMed Google Scholar). Although the accumulation of β-catenin is not sufficient for signaling (25Guger K.A. Gumbiner B.M. Dev. Biol. 2000; 223: 441-448Crossref PubMed Scopus (58) Google Scholar, 26Staal F.J. Noort Mv M. Strous G.J. Clevers H.C. EMBO Rep. 2002; 3: 63-68Crossref PubMed Scopus (286) Google Scholar), it is a key feature of Wnt activation. Given the number and variety of components that can impact β-catenin signaling at the level of the axin-scaffold phospho-destruction complex (for review, see Ref. 20Daugherty R.L. Gottardi C.J. Physiology. 2007; 22: 303-309Crossref PubMed Scopus (208) Google Scholar), knowing the intrinsic stabilizing features of β-catenin will be important for understanding how these various factors cross-regulate β-catenin signaling. A previous study found that forms of β-catenin lacking the C-terminal domain (CTD) failed to accumulate in flies, even during Wnt signaling (27Cox R.T. Pai L.M. Kirkpatrick C. Stein J. Peifer M. Genetics. 1999; 153: 319-332PubMed Google Scholar). Recent evidence indicates that the CTD negatively impacts β-catenin binding to axin and APC using the isothermal titration calorimetry method (19Choi H.J. Huber A.H. Weis W.I. J. Biol. Chem. 2006; 281: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), but how the CTD restricts β-catenin binding to axin has remained unresolved. Although the CTD plays an established role in recruiting factors required for gene expression, most interaction partners depend on the more proximal, structured elements of this region, such as helix C, which directly follows the 12th arm repeat (28Sierra J. Yoshida T. Joazeiro C.A. Jones K.A. Genes Dev. 2006; 20: 586-600Crossref PubMed Scopus (333) Google Scholar, 29Xing Y. Takemaru K. Liu J. Berndt J.D. Zheng J.J. Moon R.T. Xu W. Structure. 2008; 16: 478-487Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Functions for the more distal, unstructured region of the CTD remain poorly defined despite its contribution to β-catenin nuclear signaling (27Cox R.T. Pai L.M. Kirkpatrick C. Stein J. Peifer M. Genetics. 1999; 153: 319-332PubMed Google Scholar). The following study demonstrates that the CTD is required for the accumulation of β-catenin through shielding β-catenin from the axin-scaffold phospho-destruction complex. YFP cDNA was amplified by PCR from pEYFP vector (Clontech) and subcloned into BamHI and XbaI restriction sites of pECFP-C1 (Clontech) vector to generate pECFP-EYFP plasmid. β-Catenin fragments were amplified by PCR and cloned into XhoI and BamHI restriction sites of the pECFP-EYFP plasmid. Coding sequences of β-catenin amino acids 140–663 and ICAT were fused using PCR and also cloned into the XhoI and BamHI restriction sites of pECFP-EYFP. The coding sequence of peptide linker GGGGSGGGGS or PPPPPPPPPP was introduced into overlapping primer regions between β-catenin and ICAT. N-terminal FLAG-tagged full-length β-catenin and S33YD695 were kind gifts of Dr. Eric Fearon (University of Michigan). FLAG-tagged β-catenin truncations were amplified from FLAG-tagged full-length β-catenin constructs and cloned into pCDNA 3 vector (Invitrogen). β-Catenin amino acids 663–666 were changed to 4 prolines using the QuikChange site-directed mutagenesis kit (Stratagene). To introduce alanine mutations into β-catenin residues 733–759 and 760–773, corresponding DNA sequences for the alanine stretches were synthesized (IDT), annealed in vitro, and PCR-fused into β-catenin fragments. Coding sequence of β-catenin armadillo repeats (amino acids 140–663) was amplified by PCR and inserted into BamHI and XbaI restriction sites of pm vector (Clontech). Coding sequences of β-catenin CTD (amino acids 664–781) and ICAT were also amplified using PCR and cloned into BamHI and XbaI restriction sites of pVP16 vector (Clontech). The coding sequence of β-catenin amino acids 520–781 was PCR-amplified and inserted into HindIII and XbaI restriction sites of p3xFLAG-CMV-10 vector (Sigma). pCS2+ Myc-Zebrafish β-catenin 1 (zβ-cat-1) and pCS2+ MycZebrafish β-catenin 2 (zβ-cat-2) plasmids are described in Bellipanni et al. (30Bellipanni G. Varga M. Maegawa S. Imai Y. Kelly C. Myers A.P. Chu F. Talbot W.S. Weinberg E.S. Development. 2006; 133: 1299-1309Crossref PubMed Scopus (115) Google Scholar). GST-ICAT, FLAG-ICAT, and Myc-Xenopus C-cadherin cyto-domain are described in Gottardi et al. (31Gottardi C.J. Gumbiner B.M. Am. J. Physiol. Cell Physiol. 2004; 286: C747-CC756Crossref PubMed Scopus (66) Google Scholar). GST-axin-catenin binding domain (CBD) (amino acids 436–498) and His-β59 (β-catenin arm repeats 1∼12) were kindly provided by William Weis (Stanford University), and GST-β-catenin CTD (amino acids 695∼781) was provided by García de Herreros (Universitat Autònoma de Barcelona). GST fusion proteins were expressed in BL-21 cells (GE Healthcare) and purified using glutathione-Sepharose beads 4B (GE Healthcare) according to standard methods. The following antibodies were used for Western blots: mouse monoclonal anti-FLAG (Sigma, F3165), mouse monoclonal anti-β-catenin clone 14 (BD Transduction Laboratories), rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, SC-25778), mouse monoclonal anti-c-Myc clone 9E10 (Sigma), anti-RGS-His (Qiagen), rabbit polyclonal anti-ICAT (31Gottardi C.J. Gumbiner B.M. Am. J. Physiol. Cell Physiol. 2004; 286: C747-CC756Crossref PubMed Scopus (66) Google Scholar), and goat anti-mouse, anti-rabbit, and rabbit anti-goat IgG-horseradish peroxidase secondary antibodies (Bio-Rad). Cells were lysed in standard 1% Triton X-100 lysis buffer (20 mm Tris pH 7.5, 1%Triton X-100, 150 mm NaCl, 5 mm EDTA, 10% glycerol) containing protease inhibitors (Roche Applied Science). Protein concentrations were measured using the Bradford reagent (Bio-Rad). For immunoprecipitation and affinity precipitations, protein samples were incubated with a 1:200 ratio of specific antibody or GST fusion proteins for 2 h at 4 °C followed by 4 washes in 1% Triton X-100 lysis buffer, one wash in 0.1% Triton X-100 lysis buffer (same composition as 1% Triton X-100 lysis buffer except with 0.1% Triton X-100), and denatured by heating in SDS protein loading buffer. Proteins were separated on SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted. Immunoblots were developed in ECL solution (GE Healthcare) and exposed to Hyperfilm-ECL (GE Healthcare). 1 × 106 cells per well of HEK293T cells were seeded into 6-well dishes and transiently transfected with 1.0 μg of various FLAG-tagged β-catenin constructs. 24 h after transfection, cells were split into equal parts and cultured another 24 h before treatment with 20 μg/ml cycloheximide. Cells were washed with ice-cold phosphate-buffered saline at 0-, 1-, 2-, 4- and 6-h time points and lysed in 1% Triton X-100 lysis buffer. Protein concentrations were measured by the Bradford assay. Equivalent protein amounts were separated on 8% SDS-PAGE. Western blots were performed using anti-FLAG and anti-GAPDH antibody. ECL Western blot films were scanned, and ImageJ software was used for quantification. Thresholds were set to eliminate the background, and the integrated densities were calculated. Ectopic expressed β-catenin levels obtained from anti-FLAG immunoblots were normalized to GAPDH levels obtained from the same blot re-probed with anti-GAPDH antibody. Protein levels at different time points were normalized to the 0-h time points, and protein turnover rates were shown in percentage remaining relative to the 0-h time points. Experiments were performed at least three times, and the final results were shown as the mean ± S.D. For [35S]methionine/cysteine metabolic labeling and pulse-chase experiments, transiently transfected Cos-7 cells were incubated in methionine/cysteine-free Dulbecco's modified Eagle's medium for 30 min at 37 °C and subsequently labeled with 0.1mCi/ml PerkinElmer Life Sciences protein labeling mix (NEG772007MC) for 20 min at 37 °C. Cells were lysed in 1% Triton X-100 lysis buffer at various time points, and equal amounts of proteins were incubated with 10 μg of GST-ICAT for 2 h at 4 °C. Sepharose beads were washed 4 times with 1% Triton X-100 lysis buffer and once with 0.1% Triton X-100 buffer and boiled in SDS protein loading buffer. Protein samples were separated on SDS-PAGE, dried, and subjected to autoradiography and phosphorimage analysis (FujiFilm FLA-5100 Imager). Cos7 cells were plated at 1 × 106 cells per well and transfected accordingly. After 36 h, cells were washed twice with phosphate-buffered saline and incubated in phosphate-free labeling media for 30 min. Cells were then labeled with 120 μCi of [32P]orthophosphate (PerkinElmer Life Sciences) in 2 ml of labeling media for 3 h, washed with phosphate-buffered saline twice, and lysed in 1% Triton X-100 lysis buffer. Immunoprecipitations were performed using anti-FLAG antibody, separated on Criterion pre-cast gels (Bio-Rad), and subjected to autoradiography. Western blot was performed on ⅓ of each sample to confirm immunoprecipitation efficiency. A mammalian two-hybrid assay was performed in HEK293T cells according to Mammalian Matchmaker Two-hybrid Assay kit (Clontech) manual. Briefly, 0.1 μg of pM-arm (amino acid 140–663) and 0.1 μg of pVP16-CTD were co-transfected together with 0.3 μg GAL4-Luc reporter and 0.1 μg of Renilla luciferase plasmid. Cells were lysed 36 h after transfection, and luciferase activities were measured on a microplate dual-injector luminometer (Veritas) using the Dual-luciferase Assay kit (Promega). All transfections were performed in triplicate, and data are expressed as the mean ± S.D. Cos-7 cells were transfected with the FRET constructs in 3.5-mm glass-bottom dishes (MatTek Corp.). After 48 h, acceptor photobleaching FRET was performed at the Northwestern University Cell Imaging Facility on a Zeiss LSM510 META system (Carl Zeiss Inc.) equipped with a 63 × 1.4 Plan-Apochromat oil-immersion objective. An argon laser was used to excite CFP and YFP at 458-nm wavelength, and emission signals were recorded in META detector λ detection mode. YFP signals of cells were bleached using a 514-nm laser line at 100% power for 100 iterations, and time series images were captured for both before and after bleach. FRET efficiency was calculated using the equation EFRET = ([CFPafter − CFPbefore] × YFPbefore)/([YFPbefore − YFPafter] × CFPafter) to normalize for the amount of YFP bleached, where CFPbefore and YFPbefore are the mean pre-bleach fluorescence intensities of CFP and YFP, respectively, and CFPafter and YFPafter are the mean post-bleach fluorescence intensities of CFP and YFP. Multiple cells from at least three different transfections were measured for each construct. A previous study found that β-catenin lacking its C terminus failed to accumulate in flies (27Cox R.T. Pai L.M. Kirkpatrick C. Stein J. Peifer M. Genetics. 1999; 153: 319-332PubMed Google Scholar). To determine whether this reduced accumulation was because of enhanced protein turnover, we generated a panel of truncation mutants (Fig. 1A) and analyzed their half-lives using the cycloheximide chase method. β-Catenin lacking the entire C-terminal region distal to the 12th arm-repeat (amino acids 664–781, Δ664) is degraded more quickly than the full-length protein (Fig. 1, B and C). Similarly, a truncation mutant that retains Helix C (a structured region that “caps” the end of 12th arm repeat) and lacks only the unstructured region of the β-catenin C terminus (29Xing Y. Takemaru K. Liu J. Berndt J.D. Zheng J.J. Moon R.T. Xu W. Structure. 2008; 16: 478-487Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) displays a turnover rate comparable with the Δ664 mutant (Fig. 1D, Δ695 t½ = ∼2 h). Importantly, the instability of Δ695 can be fully rescued by preventing phosphorylation at serine 33 by GSK3β, which is known to comprise a recognition site for the SCFβ-TrCP ubiquitin ligase (3Hart M. Concordet J.P. Lassot I. Albert I. del los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Benarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar) (Fig. 1D; S33Y Δ695). Of note, the turnover of Δ695 is similar to the Δ664 mutant, and S33Y Δ695 is similar to full-length β-catenin. These data suggest that truncating the C terminus does not generate a grossly misfolded protein that is unstable but, rather, indicates that the flexible C-terminal region of β-catenin is intrinsically required for its stabilization. To understand how the C terminus promotes β-catenin stabilization, we first asked whether the C terminus could bind a factor required for β-catenin stability. Although the extreme C terminus of β-catenin contains a highly conserved PDZ binding domain motif, removing this domain had no obvious effects on β-catenin turnover compared with the full-length protein (Fig. 1, B and C). This suggests that the PDZ binding domain motif and its various potential binding partners (32Kanamori M. Sandy P. Marzinotto S. Benetti R. Kai C. Hayashizaki Y. Schneider C. Suzuki H. J. Biol. Chem. 2003; 278: 38758-38764Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 33Nishimura W. Yao I. Iida J. Tanaka N. Hata Y. J. Neurosci. 2002; 22: 757-765Crossref PubMed Google Scholar, 34Perego C. Vanoni C. Massari S. Longhi R. Pietrini G. EMBO J. 2000; 19: 3978-3989Crossref PubMed Scopus (104) Google Scholar, 35Dobrosotskaya I.Y. James G.L. Biochem. Biophys. Res. Commun. 2000; 270: 903-909Crossref PubMed Scopus (140) Google Scholar) are dispensable for β-catenin stabilization. To rule out the possibility that other regions of the C terminus interact with a factor required for stabilization (Fig. 1E, schematic), we overexpressed the β-catenin CTD or arm repeats 10–12 and the CTD (arm10CTD) in HEK293T cells and looked for a reduction in the levels of endogenous cytosolic β-catenin. Cytosolic β-catenin can be selectively affinity-precipitated with the β-catenin binding partner, ICAT (GST-ICAT). ICAT is an 81-amino acid polypeptide that binds to the cadherin-free, cytosolic pool of β-catenin and restricts its access to TCFs and transcriptional activators (36Tago K. Nakamura T. Nishita M. Hyodo J. Nagai S. Murata Y. Adachi S. Ohwada S. Morishita Y. Shibuya H. Akiyama T. Genes Dev. 2000; 14: 1741-1749PubMed Google Scholar, 37Daniels D.L. Weis W.I. Mol. Cell. 2002; 10: 573-584Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We have previously established that the GST-ICAT precipitable pool of β-catenin corresponds to the Wnt-activated signaling pool typically assessed by standard detergent-free fractionation methods (31Gottardi C.J. Gumbiner B.M. Am. J. Physiol. Cell Physiol. 2004; 286: C747-CC756Crossref PubMed Scopus (66) Google Scholar, 38Maher M.T. Flozak A.S. Stocker A.M. Chenn A. Gottardi C.J. J. Cell Biol. 2009; 186: 219-228Crossref PubMed Scopus (104) Google Scholar). Thus, this simple pulldown method can reflect changes in abundance of the cytosolic signaling pool of β-catenin. As seen in Fig. 1E, neither CTD construct reduced the accumulation of cytosolic β-catenin, as would be predicted if the C terminus sequestered a factor required for β-catenin stabilization. These CTD constructs did not affect base-line cytosolic levels of β-catenin (NaCl, control treated) or “activated” β-catenin using the Wnt pathway agonist and GSK3 inhibitor, lithium chloride (LiCl). Furthermore, overexpression of the C-terminal domain appears insufficient to drive interactions with binding partners that can be discerned by immuno- and affinity-precipitation analyses (supplemental Fig. S1 and Ref. 28Sierra J. Yoshida T. Joazeiro C.A. Jones K.A. Genes Dev. 2006; 20: 586-600Crossref PubMed Scopus (333) Google Scholar). Because the C terminus of β-catenin has been found to bind the central armadillo repeat region of β-catenin in some assays (27Cox R.T. Pai L.M. Kirkpatrick C. Stein J. Peifer M. Genetics. 1999; 153: 319-332PubMed Google Scholar, 39Castaño J. Raurell I. Piedra J.A. Miravet S. Duñach M. García de Herreros A. J. Biol. Chem. 2002; 277: 31541-31550Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and full-length β-catenin exhibits a lower affinity for axin than β-catenin lacking the CTD (19Choi H.J. Huber A.H. Weis W.I. J. Biol. Chem. 2006; 281: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), we asked whether the C terminus of β-catenin could antagonize the destruction of β-catenin by directly interacting with the arm repeat region of β-catenin and competing arm-repeat binding to axin. In agreement with previous studies (19Choi H.J. Huber A.H. Weis W.I. J. Biol. Chem. 2006; 281: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 39Castaño J. Raurell I. Piedra J.A. Miravet S. Duñach M. García de Herreros A. J. Biol. Chem. 2002; 277: 31541-31550Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), the arm repeat region lacking the flanking N- and C-terminal regions (His-arm; amino acids 140–663) can be affinity-precipitated by the CBD of axin (GST-axin-CBD; amino acids 436–498) ∼8 times more efficiently than the full-length β-catenin (His-β-catenin; Fig. 2A). To determine whether this difference in binding is mediated through the flexible C-terminal region of β-catenin, we incubated the arm-repeat region and the GST-axin-CBD in the presence of increasing amounts of the β-catenin CTD (residues 695–781). At high molar excesses, the β-catenin CTD polypeptide can interfere with axin binding to the arm repeat region, whereas an equivalent molar excess of GST protein shows little effect (Fig. 2B). These data raise the possibility that the CTD of β-catenin promotes β-catenin stabilization by directly interfering with the axin/β-catenin binding interaction. In Drosophila the C-terminal region of Armadillo was found to physically interact with the central arm repeat domain in a yeast two-hybrid screen (27Cox R.T. Pai L.M. Kirkpatrick C. Stein J. Peifer M. Genetics. 1999; 153: 319-332PubMed Google Scholar). Because direct “trans” binding between the CTD and arm-repeat domain has been difficult to observe using in vitro binding assays (19Choi H.J. Huber A.H. Weis W.I. J. Biol. Chem. 2006; 281: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), we sought to determine whether this interaction could be confirmed using a mammalian two-hybrid assay" @default.
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• W1987486570 title "The Terminal Region of β-Catenin Promotes Stability by Shielding the Armadillo Repeats from the Axin-scaffold Destruction Complex" @default.
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