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Systematic Peptide Array-based Delineation of the Differential β-Catenin Interaction with Tcf4, E-Cadherin, and Adenomatous Polyposis Coli

2005; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m410215200

1083-351X

Robert Gail, Ronald Frank, Alfred Wittinghofer,

RNA Research and Splicing

Nuclear accumulation of the complex between β-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on β-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective β-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the β-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site. Nuclear accumulation of the complex between β-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on β-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective β-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the β-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site. In humans there are around 35,000 protein-coding genes (1Consortium International Human Genome Sequencing Nature. 2001; 409: 860-921Crossref PubMed Scopus (17495) Google Scholar, 2Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. Zhang Q. Kodira C.D. Zheng X.H. Chen L. Skupski M. Subramanian G. Thomas P.D. Zhang J. Gabor Miklos G.L. Nelson C. Broder S. Clark A.G. Nadeau J. McKusick V.A. Zinder N. 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Gire H. Glanowski S. Glasser K. Glodek A. Gorokhov M. Graham K. Gropman B. Harris M. Heil J. Henderson S. Hoover J. Jennings D. Jordan C. Jordan J. Kasha J. Kagan L. Kraft C. Levitsky A. Lewis M. Liu X. Lopez J. Ma D. Majoros W. McDaniel J. Murphy S. Newman M. Nguyen T. Nguyen N. Nodell M. Pan S. Peck J. Peterson M. Rowe W. Sanders R. Scott J. Simpson M. Smith T. Sprague A. Stockwell T. Turner R. Venter E. Wang M. Wen M. Wu D. Wu M. Xia A. Zandieh A. Zhu X. Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10468) Google Scholar). Alternative splicing and posttranslational modifications further increase the number of different proteins. To understand their function, it is necessary to know the interactions they are involved in. In yeast, with its ∼6000 genes (3Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnston M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Science. 1996; 274 (563-567): 546Crossref PubMed Scopus (3208) Google Scholar), different high-throughput methods have defined a total of around 80,000 protein-protein interactions, with the real number probably being somewhat lower (4von Mering C. Krause R. Snel B. Cornell M. Oliver S.G. Fields S. Bork P. Nature. 2002; 417: 399-403Crossref PubMed Scopus (1906) Google Scholar). These searches have also shown that most of the proteins bind multiple other proteins. This may be coupled to diverse and sometimes opposing biological effects. Many interactions are involved in signal transduction pathways leading to differentiation, growth, or apoptosis.However, therapeutic intervention with such complexes has proven to be challenging because protein-protein interfaces are rather large and often flat, such that they are difficult to disrupt with small molecules. However, in some cases, it has been shown that this is possible (5Tilley J.W. Chen L. Fry D.C. Emerson S.D. Powers G.D. Biondi D. 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Even though a growing number of complex structures are available, the relation between interface features and binding strength is not well understood from a theoretical point of view. No parameter observable in crystal structures is correlated to its importance for the interaction. On the other hand, empirical analysis of interfaces has shown that generally only a few residues, the so-called hot spots, contribute the bulk of the binding free energy (9Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1772) Google Scholar, 10Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1621) Google Scholar). A detailed understanding of protein-protein interfaces and the rapid identification of hot spot residues are thus required to understand the thermodynamics and specificity of interactions and to apply such knowledge for the development of specific and potent inhibitory drugs.β-Catenin is an important paradigm for those studies. It exists in two distinct cellular pools, where it carries out two different functions through its binding to a number of diverse proteins. For its role in adherens junctions at the cell membrane, it interacts with the cytoplasmic tail of E-cadherin and with α-catenin. This is crucial for stable cell adhesion (11Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (685) Google Scholar). The cytoplasmic β-catenin pool serves as a signaling molecule in the Wnt pathway, which plays an important role in development and tissue maintenance and is often found deregulated in cancers (12Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar, 13Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar). In the absence of an extracellular Wnt signal, the cytoplasmic β-catenin concentration is kept low through proteolysis. For β-catenin degradation, its interaction with APC 1The abbreviations used are: APC, adenomatous polyposis coli; Tcf, T-cell factor; CBD, catenin-binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ITC, isothermal titration calorimetry. 1The abbreviations used are: APC, adenomatous polyposis coli; Tcf, T-cell factor; CBD, catenin-binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ITC, isothermal titration calorimetry. in a multiprotein complex is essential (14Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1137) Google Scholar). Wnt stimulation results in stabilization of β-catenin, which then translocates into the nucleus, where its complex with Tcf proteins (Tcf1, LEF1, Tcf3, and Tcf4) transcriptionally activates a number of target genes (15Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar, 16Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1601) Google Scholar).β-Catenin accumulates in the nucleus of most colorectal tumors and certain other cancers. This is due to mutation in either β-catenin itself or in negative regulators such as APC (17Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). The β-catenin·Tcf4 complex plays a central role in oncogenesis (18Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar), and its inhibition by dominant negative Tcf4 stops proliferation of colorectal cancer cells and induces differentiation (19van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. Tjon-PonFong M. Moerer P. van den Born M. Soete G. Pals S. Eilers M. Medema R. Clevers H. Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar), proving that β-catenin is an important anti-cancer target (20Kim J.S. Crooks H. Foxworth A. Waldman T. Mol. Cancer Ther. 2002; 1: 1355-1359PubMed Google Scholar). Recently, compounds inhibiting the association between β-catenin and Tcf4 have been described (7Lepourcelet M. Chen Y.N. France D.S. Wang H. Crews P. Petersen F. Bruseo C. Wood A.W. Shivdasani R.A. Cancer Cell. 2004; 5: 91-102Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). Targeting β-catenin implies another level of complexity due to its interactions with E-cadherin and APC, which should remain unaffected by potential drugs. Loss of the E-cadherin interaction is causal in the transition from adenomas to invasive carcinomas (21Perl A.K. Wilgenbus P. Dahl U. Semb H. Christofori G. Nature. 1998; 392: 190-193Crossref PubMed Scopus (1185) Google Scholar), so that its inhibition might actually promote tumor metastasis. Because the β-catenin·APC interaction regulates the level of β-catenin in healthy cells and its localization (22Henderson B.R. Fagotto F. EMBO Rep. 2002; 3: 834-839Crossref PubMed Scopus (240) Google Scholar), interfering with it might cause inappropriate activation of β-catenin signaling. The structures of β-catenin complexes with Tcf (23Graham T.A. Weaver C. Mao F. Kimelman D. Xu W.Q. Cell. 2000; 103: 885-896Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 24Graham T.A. Ferkey D.M. Mao F. Kimelman D. Xu W.Q. Nat. Struct. Biol. 2001; 8: 1048-1052Crossref PubMed Scopus (154) Google Scholar, 25Poy F. Lepourcelet M. Shivdasani R.A. Eck M.J. Nat. Struct. Biol. 2001; 8: 1053-1057Crossref PubMed Scopus (154) Google Scholar), E-cadherin (26Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar), and APC (27Spink K.E. Fridman S.G. Weis W.I. EMBO J. 2001; 20: 6203-6212Crossref PubMed Scopus (110) Google Scholar) have shown that these proteins all bind to the superhelical groove in the central armadillo repeat region of β-catenin and have partially overlapping binding sites, so that selectivity might be difficult to achieve (Fig. 1). The molecules described by Lepourcelet et al. (7Lepourcelet M. Chen Y.N. France D.S. Wang H. Crews P. Petersen F. Bruseo C. Wood A.W. Shivdasani R.A. Cancer Cell. 2004; 5: 91-102Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar) leave the E-cadherin interaction undisturbed but inhibit APC binding, which indicates they need to be optimized. To accomplish this goal, it is necessary to know the specific and differential binding determinants of the respective interactions.For β-catenin, hot spots have been defined by introducing alanine mutations in the armadillo domain and measuring the effect on LEF-1, APC, and conductin binding (28von 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 (166) Google Scholar). This has identified residues that are equally or differentially important for these interactions. Similarly, hot spots were defined in the catenin-binding domain (CBD) of Tcf (29Fasolini M. Wu X. Flocco M. Trosset J.Y. Oppermann U. Knapp S. J. Biol. Chem. 2003; 278: 21092-21098Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Omer C.A. Miller P.J. Diehl R.E. Kral A.M. Biochem. Biophys. Res. Commun. 1999; 256: 584-590Crossref PubMed Scopus (61) Google Scholar, 31Knapp S. Zamai M. Volpi D. Nardese V. Avanzi N. Breton J. Plyte S. Flocco M. Marconi M. Isacchi A. Caiolfa V.R. J. Mol. Biol. 2001; 306: 1179-1189Crossref PubMed Scopus (51) Google Scholar), which is located in the first 50–60 residues (32Roose J. Clevers H. Biochim. Biophys. Acta. 1999; 1424: M23-M37PubMed Google Scholar).We have analyzed β-catenin binding of Tcf4, E-cadherin, and APC in a systematic and comprehensive manner using peptide arrays and fluorescence-labeled β-catenin. The method allowed us to quickly identify specific and overlapping hot spots for different protein-protein interactions. Peptide array data could be directly correlated to affinity by equilibrium measurements using fluorescence polarization titration. We also further narrowed down the minimal binding fragment of Tcf4 and defined binding regions of E-cadherin. Surprisingly, the peptide array technology also showed that β-catenin binding of APC is not strictly defined by or restricted to its 15-mer and 20-mer repeats and allowed us to more rigorously define β-catenin binding regions in APC.During preparation of the manuscript, our conclusions about binding motifs in APC were corroborated by x-ray structural studies (33Ha 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 (257) Google Scholar, 34Xing 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 (137) Google Scholar).EXPERIMENTAL PROCEDURESExpression and Purification of β-Catenin, Tcf4, and E-Cadherin— Recombinant protein expression was done in Escherichia coli BL21(DE3) using the vector pGEX-4T1 and TB medium supplemented with 100 mg/liter ampicillin. After induction with 0.3 mm isopropyl 1-thio-β-d-galactopyranoside, the temperature was lowered from 37 °C to 18 °C for overnight production of the GST fusion proteins. The bacteria were lysed in a Microfluidizer M-110S (Microfluidics, Newton, MA). For human β-catenin, the armadillo domain (residues 151–666) was used. The buffer was 30 mm Tris/HCl, pH 8, 300 mm NaCl, and 3 mm dithioerythritol. For cell lysis, 1 mm phenylmethylsulfonyl fluoride was added. After affinity chromatography on reduced glutathione-Sepharose (Amersham Biosciences), β-catenin was cleaved off the column by thrombin and further purified by size exclusion chromatography on Superdex 75 (Amersham Biosciences). For fluorescent β-catenin (bcat-GFP), a GST-β-catenin-(151–666)-enhanced GFP fusion protein was produced as described above, and bcat-GFP cleaved off the reduced glutathione column was used directly. A Cys(Ser-Gly)3 linker was engineered to the N terminus of the human Tcf4 CBD (residues 1–53) for fluorescence labeling. Point mutations were introduced by QuikChange (Stratagene, Amsterdam, the Netherlands). The buffer was 30 mm Tris/HCl, pH 8, 200 mm NaCl, and 3 mm dithioerythritol. For lysis, 5 mm EDTA and 1 mm phenylmethylsulfonyl fluoride were added. The proteins were purified by reduced glutathione affinity columns, thrombin cleavage of the GST tag on the column, and size exclusion chromatography on Superdex 75. Two constructs of mouse Tcf4, amino acids 652–700 and 628–723, were used. Point mutations and an N-terminal linker were introduced as described for E-cadherin. The buffer was 30 mm Tris/HCl, pH 7.5, 200 mm NaCl, and 3 mm dithioerythritol. For lysis, 5 mm EDTA and 1 mm phenylmethylsulfonyl fluoride were added. Purification was done as described for Tcf4.Synthesis of Peptide Arrays—Membranes with peptide arrays were synthesized by the SPOT technique (35Frank R. Overwin H. Methods Mol. Biol. 1996; 66: 149-169PubMed Google Scholar) using an ASP222 automated SPOT synthesizer (Intavis Bioanalytic Instruments AG, Cologne, Germany). Briefly, acid-resistant ACS01 amino-pegylated cellulose membranes (0.6 μmol amino groups/cm2; AIMS Scientific Products, Braunschweig, Germany) were derivatized in a grid of spots of 0.3 cm in diameter, defining the location of the peptides by applying to each 0.1 μl of a solution containing 0.03 m Fmoc-β-alanine and 0.27 mN-acetylalanine, both as in situ formed 1-hydroxybenzotriazole esters. After 2 h of reaction while covered with glass plates, the intermediate membrane parts were blocked with 2% acetic anhydride in N,N-dimethylformamide overnight. Throughout solid phase synthesis, Fmoc-amino acid 1-hydroxybenzotriazole esters were used. After every coupling round, unreacted amino groups were blocked with acetic anhydride, Fmoc-protection was cleaved with 20% piperidine in N,N-dimethylformamide, and the terminal amino functions of the growing peptides were stained with bromphenol blue. Finally, all peptides were acetylated at their N termini, and the side chain protecting groups were cleaved by treatment with 82% trifluoroacetic acid, 3% triisobutylsilane, 5% dichloromethane, and 10% water for 16 h.Investigation of β-Catenin Binding to Peptide Arrays—After moisturization with ethanol, peptide membranes were washed three times with Tris-buffered saline (50 mm Tris/HCl, pH 8, 137 mm NaCl, and 2.7 mm KCl) and blocked overnight with 80% T-TBS (Tris-buffered saline + 0.05% Tween 20), 20% Blocking Buffer (Genosys, Deisenhofen, Germany), and 5% sucrose. After washing with T-TBS, 1 μm bcat-GFP in 24 mm Tris/HCl, pH 8, 240 mm NaCl, 3 mm dithioerythritol, 20% Genosys Blocking Buffer, and 0.05% Tween 20 was added for 2 h at room temperature. After three washing steps with T-TBS, the amount of bound bcat-GFP was quantified on a LA-5000 imager (Fujifilm, Duesseldorf, Germany) using an excitation wavelength of 473 nm and a 510-nm high pass filter for the emitted fluorescence light.Kd Determinations—Exchange of Tcf4 and E-cadherin into a degassed buffer (30 mm Tris/HCl, 200 mm NaCl, and 10 mm ascorbic acid) was performed by ultrafiltration. For fluorescence labeling, a protein concentration of 100 μm and a 3–5-fold excess of a thiol-reactive fluorophore were used. After overnight incubation at 4 °C, the excess fluorophore was removed by buffer exchange to 30 mm Tris/HCl, pH 7.5, 200 mm NaCl, and 3 mm dithioerythritol. Fluorescence polarization equilibrium titrations were performed in a FluoroMax II spectrometer (Spex Instruments, Grasbrunn, Germany) using 20–200 nm fluorescent protein and adding β-catenin-(151–666). The excitation/emission wave-lengths were 491/513, 494/516, and 346/449 nm for fluorescein, Alexa-488 (Molecular Probes, Leiden, the Netherlands), and 7-amino-4-methylcoumarin-3-acetic acid, respectively. Isothermal titration calorimetry was performed in a MicroCal instrument. A 100 μm solution of the Tcf4-(1–53) peptides was titrated to a 10 μm solution of β-catenin-(151–666). Both binding partners were in the same buffer as described above, plus 10% glycerol and 0.005% Tween 20. Fitting was done with the MicroCal software package using a one-site binding model.RESULTSDesign and Preparation of Peptide Arrays—In order to get a comprehensive view of the energetics and specificity of the different β-catenin interactions with Tcf4, E-cadherin, and APC, we applied a systematic single amino acid replacement strategy. Synthetic peptide arrays as available by the SPOT technique (36Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (917) Google Scholar) can easily be assembled in parallel on a microscale. The longest peptide successfully synthesized thus far by this technique consists of 44 residues (37Toepert F. Pires J.R. Landgraf C. Oschkinat H. Schneider-Mergener J. Angew. Chem. Int. Ed. Engl. 2001; 40: 897-900Crossref PubMed Scopus (45) Google Scholar). Because the CBD of Tcf contains an additional 9 residues, its synthesis is, by itself, challenging. However, because the respective CBDs are unstructured (27Spink K.E. Fridman S.G. Weis W.I. EMBO J. 2001; 20: 6203-6212Crossref PubMed Scopus (110) Google Scholar, 31Knapp S. Zamai M. Volpi D. Nardese V. Avanzi N. Breton J. Plyte S. Flocco M. Marconi M. Isacchi A. Caiolfa V.R. J. Mol. Biol. 2001; 306: 1179-1189Crossref PubMed Scopus (51) Google Scholar, 38Huber A.H. Stewart D.B. Laurents D.V. Nelson W.J. Weis W.I. J. Biol. Chem. 2001; 276: 12301-12309Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), we reasoned that their synthesis and presentation in a binding assay should not be complicated by secondary structure and folding problems. We were therefore confident in extending the synthetic aim to longer sequences.By using the peptide companion software (www.5z.com/csps/), we predicted the absence of difficult synthetic steps. To avoid inhibitory molecular crowding due to the large molecular weight of the product, we found it necessary to reduce the concentration of starting amino functions on the membrane by blending the Fmoc-β-alanine with a 10-fold excess of the elongation-incapable N-acetyl-alanine (data not shown). The completeness of the coupling reaction was assured by repeating the spotting procedure up to four times, if necessary. Additionally, we have chosen a new, acid-resistant cellulose membrane, which allows an extended treatment with high concentrations of trifluoroacetic acid and thus a complete deprotection of the peptides (39Zander N. Mol. Divers. 2004; 8: 189-195Crossref PubMed Scopus (18) Google Scholar).Alanine Scan of Tcf4 Binding to β-Catenin—Peptide arrays with individual alanine point mutants of the complete CBD of human Tcf4 (residues 1–53) (24Graham T.A. Ferkey D.M. Mao F. Kimelman D. Xu W.Q. Nat. Struct. Biol. 2001; 8: 1048-1052Crossref PubMed Scopus (154) Google Scholar) were incubated with a fusion protein of the armadillo domain of human β-catenin and enhanced GFP (bcat-GFP). The amount of bcat-GFP bound to Tcf4 was quantified with a fluorescence imager. The quantitative evaluation is shown in Fig. 2A. The error bars represent the standard deviation from results obtained with three individually synthesized peptide arrays, showing that interaction measurement by the SPOT method was quite reproducible.Fig. 2Peptide array analysis of the β-catenin·Tcf4 interaction.A, alanine scan. Arrays with all single alanine point mutants of the catenin-binding domain of human Tcf4 (residues 1–53) were incubated with a fluorescent variant of β-catenin (bcat-GFP). Binding was quantified by fluorescence imaging. All signals were normalized to the wild-type signal. , alanine mutants; ▪, negative controls (randomized sequence); □, wild-type peptides. The sequence is indicated above the bars of the alanine mutants. B, scan for a minimal Tcf4 binding domain. A membrane with overlapping 15-mer peptides covering the whole Tcf4-CBD was probed with bcat-GFP as described previously. Different-colored bars are from three different experiments and are normalized to the strongest signal.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation of Asp16 or Phe21 had the strongest effect on β-catenin binding. Replacement of either residue for Ala resulted in a signal indistinguishable from the negative controls (randomized sequence), indicating almost complete loss of β-catenin binding to Tcf4. At least 50% reduction in signal resulted from the mutations of Glu17, Leu18, Ile19, Asp23, Leu41,orLeu48. The mutations V49A and E51A in the C-terminal part of the Tcf4-CBD resulted in a >30% signal reduction. These data confirm and extend previous hot spot studies (see below), establishing peptide array analysis as a valuable method to study β-catenin interactions. In contrast, the acidic region from residue 24 to residue 29 of Tcf4, which binds an important β-catenin lysine through alternative conformations (24Graham T.A. Ferkey D.M. Mao F. Kimelman D. Xu W.Q. Nat. Struct. Biol. 2001; 8: 1048-1052Crossref PubMed Scopus (154) Google Scholar), was of only moderate importance for β-catenin binding. Surprisingly, mutation of either Gly13 or Asn15 led to a consistent 30% increase in the amount of bcat-GFP bound to the membrane spot.Solution Assay to Characterize Hot Spot Mutants—We wondered whether peptide arrays as a non-equilibrium method reflect true relative affinities. Thus, recombinant wild-type and mutant Tcf4 peptides were prepared with an additional linker and a cysteine at the N terminus, to allow coupling of a fluorophore. The affinity of the peptides was determined by titration with the armadillo domain of human β-catenin and measurement of the increase in fluorescence polarization due to formation of a large molecular weight complex. Representative titration curves are shown in Fig. 3A for the L12A and V49A mutants, and the results are summarized in Table I.Fig. 3Affinity validation by equilibrium measurements.A, representative examples for affinity measurements by fluorescence polarization of fluorescence-labeled human Tcf4-CBD variants with β-catenin at 20 °C. The values were fitted to a quadratic binding equation. B, isothermal titration calorimetry measurements of the binding of unlabeled Tcf4-CBD variants (syringe) to β-catenin (chamber) at 25 °C. Top panels, raw heating power over time; bottom panels, fit of the integrated energy values normalized for the amount of the injected protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEquilibrium dissociation constants for the β-catenin interaction with various peptidesPeptideKdFluorophoreTcf4-(1-53) wild-type0.84 ± 0.18 nmFluoresceinTcf4-(1-53) L12A0.82 ± 0.13 nmFluoresceinTcf4-(1-53) G13A0.52 ± 0.085 nmFluoresceinTcf4-(1-53) D16A1.4 ± 0.097 μmAlexa-488Tcf4-(1-53) E17A21 ± 5.6 nmFluoresceinTcf4-(1-53) L18A12 ± 2.6 nmAlexa-488Tcf4-(1-53) I19A31 ± 3.2 nmFluoresceinTcf4-(1-53) F21A220 ± 24 nmFluoresceinTcf4-(1-53) D23A1.8 ± 0.62 nmFluoresceinTcf4-(1-53) N34A0.66 ± 0.31 nmFluoresceinTcf4-(1-53) L41A250 ± 12 nmFluoresceinTcf4-(1-53) L48A220 ± 22 nmFluoresceinTcf4-(1-53) V49A7.0 ± 0.73 nmFluoresceinTcf4-(1-53) E51A2.5 ± 0.41 nmFluoresceinTcf4-(13-27)1.4 μmAMCAaAMCA, 7-amino-4-methylcoumarin-3-acetic acidTcf4-(13-25)3.0 μmAMCATcf4-(15-27)10 μmAMCAE-cadherin-(652-700) wild-type1.8 μmFluoresceinE-cadherin-(652-700) 6E100 nmFluoresceinE-cadherin-(628-723) 6E0.52 ± 0.16 nmFluoresceinE-cadherin-(628-723) 6E L661A14 ± 2.6 nmFluoresceinE-cadherin-(628-723) 6E D674A1900 ± 38 nmFluoresceinE-cadherin-(628-723) 6E L676A9.2 ± 2.8 nmFluoresceina AMCA, 7-amino-4-methylcoumarin-3-acetic acid Open table in a new tab