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• W2061170173 abstract "Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan,Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression. Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan,Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression. glycogen synthase kinase-3 T-cell factor frequently rearranged in activated T-cells GSK-3-binding protein dimethyl suberimidate GSK-3 binding region from Axin GSK-3 binding region from Frat Dictyostelium GSK-3 homologue Dictyostelium homologue of β-catenin termed Aardvark green fluorescent protein hemagglutinin signal transducers and activators of transcription Glycogen synthase kinase-3 (GSK-3)1 is a serine/threonine protein kinase that plays a key role in several signaling pathways. GSK-3 homologues have been identified in most eukaryotes including yeast and the slime mold, Dictyostelium discoideum (reviewed in Refs. 1Plyte S.E. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (330) Google Scholar and 2Kim L. Kimmel A.R. Curr. Opin. Genet. Dev. 2000; 10: 508-514Crossref PubMed Scopus (210) Google Scholar). Targets of GSK-3 include proteins involved in transcription, translation, the control of the cytoskeleton, cell cycle, and glycogen metabolism. Phosphorylation by GSK-3 is often inhibitory. For example, GSK-3 phosphorylation inhibits glycogen synthase activity. Insulin stimulation reduces GSK-3 activity and hence increases the conversion of glucose to glycogen (1Plyte S.E. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (330) Google Scholar). GSK-3 phosphorylation also targets β-catenin for degradation. This is inhibited by Wnt stimulation, and the increased concentrations of β-catenin bind and activate members of the TCF transcription factor family (3Cohen P. Parker P.J. Woodgett J.R. Czech M.P. Molecular Basis of Insulin Action. Plenum Press, New York1985Google Scholar). In addition, GSK-3 appears to play a role in regulating nuclear export both for cyclin D1 and the transcription factors, nuclear factor of activated T-cells and D. discoideum STAT protein (4Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1933Crossref PubMed Scopus (633) Google Scholar, 5Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (448) Google Scholar, 6Ginger R.S. Dalton E.C. Ryves W.J. Fukuzawa M. Williams J.G. Harwood A.J. EMBO J. 2000; 19: 5483-5491Crossref PubMed Scopus (58) Google Scholar). The diversity of substrates is reflected in the complexity and number of regulatory mechanisms that act on GSK-3. In animals, most studies have shown the activity of GSK-3 to be negatively regulated by ligands such as epidermal growth factor, Wnt, and insulin. InDictyostelium, genetic and biochemical evidence showed both positive and negative regulation of GSK-3 activity (7Ginsburg G.T. Kimmel A.R. Genes Dev. 1997; 11: 2112-2123Crossref PubMed Scopus (42) Google Scholar, 8Plyte S.E. O'Donovan E. Woodgett J.R. Harwood A.J. Development. 1999; 126: 325-333PubMed Google Scholar). Insulin signaling is the best characterized signaling pathway. Here, GSK-3 kinase activity is inhibited through phosphorylation of an inhibitory amino-terminal serine (Ser-21 in GSK-3α and Ser-9 in GSK-3β) by protein kinase B/Akt (9Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). The recent publication of the structure of human GSK-3β can explain this mechanism of GSK-3 regulation (10Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 11ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (317) Google Scholar). GSK-3β has a basic patch of amino acids in its substrate binding groove that recognizes substrates when prephosphorylated at position +4 with respect to the target residue. The phosphorylation of GSK-3 at serine 9 by regulators such as protein kinase B and protein kinase A appears to generate a pseudosubstrate that autoinhibits GSK-3 activity by competition for substrate binding (10Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 11ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (317) Google Scholar, 12Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). The mechanism of GSK-3 regulation in response to other signals is less clear. Serine phosphorylation at a site other than serine 9 has been shown in response to Wnt ligands (13Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), while tyrosine phosphorylation and activation of GSK-3 occurs in the regulation ofDictyostelium GskA in response to stimulation with extracellular cAMP (14Kim L. Liu J. Kimmel A.R. Cell. 1999; 99: 399-408Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Inhibition of GSK-3 by insulin or activated forms of protein kinase B is not sufficient for the activation of TCF-dependent transcription (15Ding V.W. Chen R.H. McCormick F. J. Biol. Chem. 2000; 275: 32475-32481Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). By contrast, small molecule inhibitors of GSK-3 are able to activate transcription (16Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar, 17Hedgepeth C.M. Conrad L.J. Zhang J. Huang H.-C. Lee V.M.Y. Klein P.S. Dev. Biol. 1997; 185: 82-91Crossref PubMed Scopus (551) Google Scholar, 18Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). This suggests that different pools of GSK-3 exist within the cell to integrate upstream signals with specific downstream targets. These independent pools could be generated by interaction with scaffolding proteins. The best understood of these is Axin, which templates GSK-3 phosphorylation of β-catenin as part of a multiprotein complex that degrades β-catenin (reviewed in Refs. 19Polakis P. Genes Dev. 2000; 14: 1837-1851PubMed Google Scholarand 20Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar). Wnt signal transduction interferes with the function of this complex, leading to the stabilization of β-catenin and the activation of β-catenin/TCF-dependent transcription. Other proteins that have been suggested to bind GSK-3 directly include Frat/GBP, presenilin, and Muc1 (21Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Mingzi Chen M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 22Takashima A. Murayama M. Murayama O. Kohno T. Honda T. Yasutake K. Nihonmatsu N. Mercken M. Yamaguchi H. Sugihara S. Wolozin B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9637-9641Crossref PubMed Scopus (397) Google Scholar, 23Hong Y.R. Chen C.H. Chang J.H. Wang S. Sy W.D. Chou C.K. Howng S.L. Biochim. Biophys. Acta. 2000; 1492: 513-516Crossref PubMed Scopus (42) Google Scholar). Frat competes with Axin for binding to GSK-3 and also interacts with the upstream Wnt signaling component Dishevelled, leading to the suggestion that it titrates GSK-3 from Axin in response to Wnt signaling (24Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (353) Google Scholar, 25Salic A. Lee E. Mayer L. Kirschner M.W. Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). In this paper, we examine the interactions of GSK-3 with itself as a homodimer and with Frat and Axin. We use the crystal structural data to generate a library of surface scanning mutants. From this library, we have identified four mutants that interact with only Axin or Frat but not both. This argues that although binding of Axin and Frat to GSK-3 may be mutually exclusive, they do not bind through an identical interaction site. To assess the importance of the Axin binding, we examine non-Axin binding GSK-3 mutants in the context of a number of organisms. As expected, Axin binding is required for the effects of dominant negative GSK-3 on patterning in Xenopusembryos. The Axin binding site motif is conserved in all GSK-3 kinase family members, and we show that it is required for GSK-3 function in the nonmetazoan, Dictyostelium. Finally, we show that Axin binding is not the only interaction able to regulate TCF-dependent gene expression. His-tagged GSK-3β was purified from baculovirus-infected insect cells as described in Ref. 10Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar. Cross-linking studies were based on a method by Prodromou et al. (26Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar). GSK-3, ATP (2 mm final concentration), the cross-linking reagent dimethyl suberimidate (DMS; 50 mmstock solution), FRATtide, and AxinGID were diluted in reaction buffer (100 mm HEPES, pH 8.0, 150 mm NaCl, 5 mm MgCl2) and made up to a total volume of 40 μl. DMS was added to the mixture at 30-fold molar excess over the primary amine content of the GSK-3. The reactions were incubated at 4 °C for 90 min. and stopped by the addition of 25 mmTris, pH 6.8, and SDS loading buffer. The mixtures were analyzed on 10 or 12% SDS-PAGE gels and stained with Coomassie Blue. [35S]Methionine-labeled GSK-3 proteins (wild type and mutant) were made using the TNT-coupledin vitro transcription/translation system according to the manufacturer's instructions (Promega). 5 μl of each mix was removed and mixed with 20 μl of loading buffer to check the efficiency of the reaction. The remainder was split in half and mixed with purified GST fusion protein and 25 μl of washed glutathione-Sepharose beads (Amersham Pharmacia Biotech). This was made up to 250 μl with ice-cold buffer (100 mm NaCl, 0.5% Nonidet P-40, 20 mm Tris-HCl, pH 8.0, 1 mm EDTA supplemented with a Complete protease inhibitor tablet (Roche Molecular Biochemicals), 1 mm phenylmethylsulfonyl fluoride and 0.5 mm dithiothreitol). Following incubation on a rotary mixer at 4 °C for 1 h, samples were pelleted, washed three times with ice-cold buffer, and analyzed by SDS-PAGE and autoradiography. Purified His-tagged GSK-3 (230 nm) was mixed with 20 μl of washed Talon® metal affinity resin beads (CLONTECH) in buffer (100 mm NaCl, 0.1% Nonidet P-40, 20 mm Tris-HCl, pH 8.0, supplemented with a Complete protease inhibitor tablet without EDTA and 1 mm phenylmethylsulfonyl fluoride) at room temperature for 20 min. After centrifugation, unbound GSK-3 was removed by washing three times with buffer. [35S]methionine-labeled in vitro translated GSK-3, AxinGID, or Frat was added to the pellet and made up to a total volume of 250 μl in buffer. The tubes were placed on a rotary mixer at 4 °C for 1 h. Following centrifugation, the beads were washed three times in ice-cold buffer. 45 μl of loading buffer was added to the pellets, and the results were analyzed by SDS-PAGE and autoradiography. 293 cells were seeded at 1 × 106 cells/10-cm dish 48 h before an experiment. Each dish was transfected with 1.2 μg of vector (pcDNA3) or 0.6 μg of GSK-3 (wild type or mutant) plus 0.6 μg of vector as described previously (36Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). Immunoprecipitations and kinase assays were carried out according to Ref. 34Ryves W.J. Fryer L. Dale T.C. Harwood A.J. Anal. Biochem. 1998; 264: 124-137Crossref PubMed Scopus (66) Google Scholar. The activities of mutant GSK-3 proteins were expressed as a percentage of transfected wild type GSK-3. HEK 293 and Madin-Darby canine kidney cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C under 5% CO2. Transfection-luciferase reporter assays and analysis of the expression of transfected constructs by Western blotting were carried out as previously described (36Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). Primary antibodies used were mouse anti-GSK-3β and mouse anti-FLAG monoclonal antibodies (Transduction Laboratories). 293 cells were seeded at 7.5 × 105 cells/10-cm dish 48 h before an experiment. Each dish was transfected with 0.4 μg of construct (HA-tagged wild type GSK-3 or GSK-3GR and FLAG Axin-(351–956) as indicated plus vector (pcDNA3) in a total of 0.8 μg as described previously (36Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). Immunoprecipitations were performed as described in Ref. 30Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar. Samples were loaded on 10 or 12% SDS-PAGE gels, blotted, and subjected to Western analysis. Cells were seeded at 30–40% confluence and were transfected with the specified vectors using Effectene Reagent (Qiagen, Crawley, West Sussex, UK) according to the manufacturer's instructions. After overnight incubation, cells were fixed with cold 4% paraformaldehyde in phosphate-buffered saline and processed for immunocytochemistry as previously described (36Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). HA epitope was detected with the rat monoclonal antibody 3F10 (Roche Molecular Biochemicals). FLAG epitope was detected with the mouse monoclonal anti-FLAG M2 (Sigma). Quantitation of expression was carried out by taking thin optical slices of transfected cells by confocal microscopy. Optical sections were selected to intersect the center of the nucleus and were quantified by density analysis using the Bio-Rad Confocal Software line intensity tool. After linearization with NotI, the mRNA expression vectors for XGSK-3 were transcribed in vitro using SP6 polymerase (Promega) in the presence RNA cap analog (New England Biolabs). The vegetal poles of single ventral blastomeres of four-cell embryos were injected with 2.5 ng of the indicated mRNA transcript and allowed to develop for 3 days. XGSK-3 parental constructs were described by Pierce and Kimelman (29Pierce S.B. Kimelman D. Development. 1995; 121: 755-765Crossref PubMed Google Scholar) and were provided by Dr. P. Klein. Mutant GSK-3 constructs were generated using site-directed mutagenesis according to the manufacturer's instructions (QuikChange; Stratagene). Surface scanning mutagenesis was performed on HA-tagged GSK-3 cDNAs within the pcDNA3.1+ expression vector. The GR mutation was introduced into XGSK-3 plasmids in a pCS2+ vector background for theXenopus studies (29Pierce S.B. Kimelman D. Development. 1995; 121: 755-765Crossref PubMed Google Scholar). The GR substitution was introduced into GskA in the plasmid pDXA-gskA (8Plyte S.E. O'Donovan E. Woodgett J.R. Harwood A.J. Development. 1999; 126: 325-333PubMed Google Scholar), which expresses thegskA cDNA from the actin15 promoter. The K208A/E209Q substitution in the GSK-3 binding domain of Frat was generated by site-directed mutagenesis of the FLAG-tagged cDNA within the pcDNA3.1+ expression vector. β-Catenin-GFP expression constructs were made by fusing GFP to the C terminus of murine β-catenin in the pEGFP vector (CLONTECH). Wild type andgskA mutant cells were grown at 22 °C in axenic medium pH 6.4. Cells were transformed by electroporation (54Howard P.K. Ahern K.G. Firtel R.A. Nucleic Acids Res. 1988; 16: 2613-2623Crossref PubMed Scopus (169) Google Scholar). Cells were observed either on SM agar or growing in axenic medium. For suspension development, cells were washed in KK2 (15.5 mmKH2PO4, 3.8 mmK2HPO4, pH 6.2) and shaken for 8 h in KK2. 1 mm cAMP was added, and cells were shaken further for 16 h (35Grimson M.J. Coates J.C. Reynolds J.P. Shipman M. Blanton R.L. Harwood A.J. Nature. 2000; 408: 727-731Crossref PubMed Scopus (116) Google Scholar). In previous work, we determined the structure of human GSK-3β and showed that it formed an intimate head-to-tail dimer (10Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). Since apparent dimers in protein crystals may be artifacts of the crystal lattice, we sought to determine whether GSK-3 formed a stable dimer in solution using chemical cross-linking. We found that GSK-3 formed dimers at a concentration of the dimethyl suberimidate cross-linking reagent that was previously used to identify intimate dimers of the hsp90 protein (26Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar). Higher molecular weight ladders of cross-linked product were not detected, suggesting that the cross-linking reaction was specifically linking soluble dimers. When GSK-3 was titrated from 10 to 1.25 μm, the ratio of dimer to unlinked product was reduced, suggesting that the affinity of GSK-3 for itself was in the low micromolar range (Fig.1A). To confirm this observation, we attempted to co-precipitate in vitrotranslated GSK-3 with His-tagged GSK-3 at a final concentration of 230 nm. Although His-tagged GSK-3 was able to associate with Frat, it was unable to associate with itself, suggesting that the affinity of GSK-3 dimer formation is significantly less than that of the GSK-3-Frat interaction (Fig. 1B). In the cross-linking assays, we observed the formation of heteromeric complexes between GSK-3 and GSK-3-binding peptides from Frat (FRATtide) (31Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (201) Google Scholar) and Axin (AxinGID) of 38 and 59 amino acids, respectively (Fig. 1,C and D). The addition of AxinGID and FRATtide strongly interfered with GSK-3-GSK-3 cross-linking, suggesting that Frat and Axin either bind to the dimer interface or allosterically alter the ability of GSK-3 to self-associate. Maximal inhibition of GSK-3-GSK-3 dimer formation was obtained at ∼1:1 molar ratios of Frat or Axin to GSK-3. The ability of the AxinGID to self-associate was investigated, since previous studies showed that full-length Axin formed multimers. Under conditions in which GST-Axin was able to associate with in vitro translated GSK-3, GST-Axin did not associate with in vitro translated Axin (Fig. 1E). The further addition of purified GSK-3 to the Axin in vitro translation mix failed to generate Axin/GST-Axin interactions, suggesting that Axin dimerization was not required for GSK-3 binding. CD spectroscopy studies of purified AxinGID indicated that this region had no structure. 2R. Dajani, unpublished observation. To identify sites on GSK-3 that mediate interactions with Axin and Frat, we generated a series of 79 point mutations predominantly at the surface of the molecule. The GSK-3 point mutants were initially screened for binding using GST-Axin and GST-Frat in a precipitation assay in vitro (Fig.2A). Mutational changes were engineered to alter the surface charge, or hydrophobicity. Most mutants bound with similar efficiency to both Axin and Frat. 60% (48/79 mutations) bound at levels indistinguishable from the wild type protein, whereas the remaining mutants showed reduced binding ranging from 0 to 75% efficiency when expressed as a percentage of the wild type (Table I; Fig. 2, B–D). The nature of the amino acid substitution was a major contributor to the level of binding. For example, V139I bound GST-AxinGID and GST-Frat with close to wild type efficiency, while V139D bound with only 5 and 10% efficiencies, respectively. Similarly, a change of serine 237 to an aspartic acid residue (S237D) partially interfered with both Axin and Frat binding, while mutation of the same residue to alanine (S237A) failed to alter binding.Table ISummary of GSK-3 mutant studiesLittle effect on bindingAA1-aAA, amino acid. No.AA changeAxin binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Frat binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Kinase activity1-cGSK-3HA mutants were transfected into HEK 293 cells, immunoprecipitated with anti-HA monoclonal antibody, and assessed for kinase activity as previously described (34).Wild type1001001009S9A100ND1-dNot detected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 but equal effects on bindingAA No.AA changeAxin binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Frat binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Kinase activity1-cGSK-3HA mutants were transfected into HEK 293 cells, immunoprecipitated with anti-HA monoclonal antibody, and assessed for kinase activity as previously described (34).Wild type10010010085K85M50098L98R1510100I100R05108N108A510101-eLow expression.113R113L5060117Y117R7070139V139D510142V142D2020156I156R4060181D181R5060206Q206E00216Y216F4040228I228K55229F229Q55237S237D5050252L252S5050285N285E2030288Y288R510332L332R101075333E333K505060343L343R000350L350I8060366E366K1520Differential effects on bindingAA No.AA changeAxin binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Frat binding1-bMeasured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.Kinase activity1-cGSK-3HA mutants were transfected into HEK 293 cells, immunoprecipitated with anti-HA monoclonal antibody, and assessed for kinase activity as previously described (34).180R180E10030260/261/263/ 267/268D260E/S261N/ V263I/V267G/E268R060267/268V267G/E268R080100293F293Q520312E312K5501-a AA, amino acid.1-b Measured as described in the legend to Fig. 2 and expressed as a percentage of wild type GSK-3.1-c GSK-3HA mutants were transfected into HEK 293 cells, immunoprecipitated with anti-HA monoclonal antibody, and assessed for kinase activity as previously described (34Ryves W.J. Fryer L. Dale T.C. Harwood A.J. Anal. Biochem. 1998; 264: 124-137Crossref PubMed Scopus (66) Google Scholar).1-d Not detected.1-e Low expression. Open table in a new tab Residue Lys85, which forms part of the ATP binding site and is commonly mutated to generate kinase-dead variants, was changed to either a methionine or an arginine (27He X. Saint-Jeannet J.-P. Woodgett J.R. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (446) Google Scholar, 28Dominguez I. Itoh K. Sokol S.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8498-8502Crossref PubMed Scopus (285) Google Scholar, 29Pierce S.B. Kimelman D. Development. 1995; 121: 755-765Crossref PubMed Google Scholar). The K85M mutant showed negligible binding to Frat and Axin. Since Lys85 is not readily accessible, it is unlikely to be directly involved in binding to regulatory proteins, and the failure of binding is probably due to the disruption of the correct folding of GSK-3. By contrast, when Lys85 was mutated to an arginine (K85R), the mutant bound with wild type efficiency to both Axin and Frat (Fig. 2A). Although the K85R mutation also generates a kinase-dead variant of GSK-3, it is a more conservative change and preserves Axin and Frat binding. This observation contradicts previous studies that suggested that GSK-3 kinase activity was required for Axin binding (30Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). Kinase activity does not correlate with binding, but it may provide an independent readout for the correct folding of the molecule. We characterized the activity of several mutants using transfection-based immunoprecipitation kinase assays (Table I). Due to variable levels of expression, the activity of many mutants was difficult to assess. Among those characterized, we found that L343R, which failed to bind Axin or Frat, was kinase-inactive. Leu343 was not present on the surface and may, like K85M, have disrupted the overall structure (Fig.2, B–D). Four mutants showed differential binding to Axin and Frat (Table I). Three of these differentially interfered with Axin binding (V267G/E268R, F293Q, and E312K), while one differentially interfered with Frat binding (R180E). Interestingly, R180E is one of three residues (Arg96, Arg180, and Lys205) that are involved in the recognition of the prephosphorylated serine/threonine in GSK-3-dependent targets ((S/T)XXX(pS/pT); where pS and pT represent phosphoserine and phosphotyrosine, respectively) (10Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). By contrast, mutation of Arg96 and Lys205 had no effect on Axin or Frat binding. With the exception of Glu312, all of the residues that differentially affected binding clustered on the GSK-3 dimer interface (Fig. 2, B–D). This supports the suggestion that binding of Frat and Axin sterically interferes with GSK-3 dimer formation. The binding of both to the same face of GSK-3 is consistent with the finding that Axin and Frat binding to GSK-3 are mutually exclusive (31Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (201) Google Scholar). The strongest differential binding observed was with the mutant GSK-3 V267G/E268R (GSK-3GR), which bound Frat efficiently but had severely reduced binding to GST-AxinGID. We chose to study the GSK-3GR further because it was kinase-active and localized close to a further differential “Frat > Axin” binding mutant (F293Q) in the crystal structure (Table I; Fig. 2C). GSK-3GR was purified from baculovirus-infected insect cells and assayed in parallel with wild type GSK-3. At similar concentrations to wild type GSK-3, GSK-3GR formed cross-linked homodimers and interacted with both Axin and Frat. In pull-down assays at lower concentrations (45 nm), the selective binding of GSK-3GR was revealed when it precipi" @default.
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• W2061170173 date "2002-01-01" @default.
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• W2061170173 title "Identification of the Axin and Frat Binding Region of Glycogen Synthase Kinase-3" @default.
• W2061170173 cites W1518972222 @default.
• W2061170173 cites W1533463539 @default.
• W2061170173 cites W1666932156 @default.
• W2061170173 cites W1833591189 @default.
• W2061170173 cites W1964859393 @default.
• W2061170173 cites W1976353556 @default.
• W2061170173 cites W1979993283 @default.
• W2061170173 cites W1982958012 @default.
• W2061170173 cites W1984704240 @default.
• W2061170173 cites W1987483436 @default.
• W2061170173 cites W1993595529 @default.
• W2061170173 cites W1994700168 @default.
• W2061170173 cites W2012039807 @default.
• W2061170173 cites W2022309233 @default.
• W2061170173 cites W2023951174 @default.
• W2061170173 cites W2029857102 @default.
• W2061170173 cites W2030557396 @default.
• W2061170173 cites W2036864436 @default.
• W2061170173 cites W2038073573 @default.
• W2061170173 cites W2039842713 @default.
• W2061170173 cites W2040401878 @default.
• W2061170173 cites W2040788683 @default.
• W2061170173 cites W2045851837 @default.
• W2061170173 cites W2046044279 @default.
• W2061170173 cites W2048675800 @default.
• W2061170173 cites W2053791137 @default.
• W2061170173 cites W2058138221 @default.
• W2061170173 cites W2066497759 @default.
• W2061170173 cites W2069704514 @default.
• W2061170173 cites W2075319861 @default.
• W2061170173 cites W2075460910 @default.
• W2061170173 cites W2078747147 @default.
• W2061170173 cites W2082387343 @default.
• W2061170173 cites W2084628289 @default.
• W2061170173 cites W2093147450 @default.
• W2061170173 cites W2094253796 @default.
• W2061170173 cites W2103171833 @default.
• W2061170173 cites W2103345757 @default.
• W2061170173 cites W2108435741 @default.
• W2061170173 cites W2119109207 @default.
• W2061170173 cites W2121745326 @default.
• W2061170173 cites W2132475598 @default.
• W2061170173 cites W2133578310 @default.
• W2061170173 cites W2141624798 @default.
• W2061170173 cites W2142493853 @default.
• W2061170173 cites W2143214449 @default.
• W2061170173 cites W2151558240 @default.
• W2061170173 cites W2154111513 @default.
• W2061170173 cites W2170134609 @default.
• W2061170173 cites W2182662973 @default.
• W2061170173 cites W2239090104 @default.
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