Title: Role of 14-3-3γ in FE65-dependent Gene Transactivation Mediated by the Amyloid β-Protein Precursor Cytoplasmic Fragment
Abstract: The amyloid β-protein precursor intracellular domain fragment (AICD) is generated from amyloid β-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3γ bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3γ bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3γ and blocked gene transactivation. 14-3-3γ required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3γ binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3γ facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3γ and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3γ modulate FE65-dependent gene transactivation. The amyloid β-protein precursor intracellular domain fragment (AICD) is generated from amyloid β-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3γ bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3γ bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3γ and blocked gene transactivation. 14-3-3γ required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3γ binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3γ facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3γ and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3γ modulate FE65-dependent gene transactivation. Amyloid β-protein precursor (APP) 3The abbreviations used are: APPamyloid β-protein precursorAICDAPP intracellular domain fragmentADAlzheimer diseaseGal4BDGal4 DNA-binding domainIPimmunoprecipitateN2aNeuro-2aWTwild typePIphosphotyrosine interactionJNKc-Jun NH2-terminal kinaseJIPJNK-interacting proteinGSTglutathione S-transferaseEGFPenhanced green fluorescent proteinNLSnuclear localization signalRNAiRNA interferenceS-richSer-rich. is thought to be a causative factor of Alzheimer disease (AD), and it is hypothesized that generation of β-amyloid from APP and its aggregation are central to the pathogenesis of AD (1Selkoe D.J. Nature. 2003; 426: 900-904Crossref PubMed Scopus (1201) Google Scholar). APP is cleaved consecutively, first at the extracellular juxtamembrane region by α- or β-secretase and second at the intramembrane region by γ-secretase (2Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5164) Google Scholar). Following the first cleavage, sAPPα or sAPPβ (large extracellular amino-terminal domains truncated at the α and β sites, respectively) is secreted, and then following the second cleavage, p3 or β-amyloid peptides are secreted, together with release of the cytoplasmic fragment into the cytoplasm. The metabolism of APP resembles that of Notch, a cell surface receptor essential for the commitment to cell differentiation (3Artavanis-Tsakonas S. Matsuno K. Fortini M.E. Science. 1995; 268: 225-232Crossref PubMed Scopus (1403) Google Scholar, 4Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1360) Google Scholar). Many type I membrane proteins, including CD44, ErbB4, neuregulin-1, and alcadein, have been found recently to be cleaved first at an extracellular juxtamembrane region and subsequently at an intramembrane region by γ-secretase (5Okamoto I. Kawano Y. Murakami D. Sasayama T. Araki N. Miki T. Wong A.J. Saya H. J. 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Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (560) Google Scholar). amyloid β-protein precursor APP intracellular domain fragment Alzheimer disease Gal4 DNA-binding domain immunoprecipitate Neuro-2a wild type phosphotyrosine interaction c-Jun NH2-terminal kinase JNK-interacting protein glutathione S-transferase enhanced green fluorescent protein nuclear localization signal RNA interference Ser-rich. The cytoplasmic domain fragment derived from APP (AICD) activates the transcription of a reporter gene in the presence of the neuron-specific adaptor protein FE65 (11Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1054) Google Scholar, 12Gao Y. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14979-14984Crossref PubMed Scopus (210) Google Scholar) and the histone acetyltransferase Tip60, but the molecular mechanisms mediating activation of transcription are not clear. Several hypotheses have been presented and remain under consideration (13Baek S.H. 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Chem. 2005; 280: 36895-36904Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Perkinton M.S. Standen C.L. Lau K.-F. Kesavapany S. Byers H.L. Ward M. McLoughlin D.M. Miller C.C.J J. Biol. Chem. 2004; 279: 22084-22091Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The AICD contains several functionally important motifs. The 653YTSI656 motif (human APP695 isoform numbering) is the basolateral sorting signal of APP in Madin-Darby canine kidney epithelial cells (20Lai A. Gibson A. Hopkins C.R. Trowbridge I.S. J. Biol. Chem. 1998; 273: 3732-3739Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The 667VTPEER672 motif contains the phosphorylation site Thr-668 and controls the stability of the overall structure of AICD (21Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 22Ramelot T.A. Gentile L.N. Nicholson L.K. 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To reveal the role of AICD in FE65-dependent gene transactivation may contribute to understanding the physiological roles of APP and the pathogenesis of AD. In the present study, we found that the 667VTPEER672 motif regulates AICD function in FE65-dependent gene transactivation through the phosphorylation of Thr-668 and interactions with the 14-3-3γ protein. Mammalian 14-3-3 proteins are a ubiquitously expressed gene family with seven distinct isoforms that are involved in various signal transduction pathways (32Aiken A. Baxter H. Dubois T. Clokie S. Mackie S. Mitchell K. Peden A. Zemlickova E. Biochem. Soc. Trans. 2002; 30: 351-360Crossref PubMed Google Scholar). 14-3-3γ is one of the most abundant isoforms in brain, skeletal muscle, and heart (33Patel Y. Martin H. Howell S. Jones D. Robinson K. Aiken A. Biochim. Biophys. Acta. 1994; 1222: 405-409Crossref PubMed Scopus (18) Google Scholar, 34Horie M. Suzuki M. Takahashi E. Tanigami A. Genomics. 1999; 60: 241-243Crossref PubMed Scopus (29) Google Scholar). We found that association of the 14-3-3γ dimer with both nonphosphorylated AICD and FE65 facilitates gene expression, whereas the phosphorylation of AICD interferes with 14-3-3γ binding and down-regulates FE65-dependent gene transactivation. These findings, taken together with our previous report that the phosphorylation of AICD suppresses its interaction with FE65 (21Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), suggest that the AICD 667VTPEER672 motif is an important regulatory region for AICD-mediated FE65-dependent gene transactivation. Plasmid Construction—cDNAs encoding the six human 14-3-3 isoforms used in this study, β (GenBank™ accession number 21328444), ϵ (GenBank™ accession number 21328449), γ (GenBank™ accession number 21464100), η (GenBank™ accession number 2146102), θ (GenBank™ accession number 2146103), and ζ (GenBank™ accession number 21735623), were amplified by PCR. The entire coding sequence was inserted into the NheI/XhoI sites of pcDNA3.1-N-FLAG to generate pcDNA3.1-FLAG-14-3-3. The cDNA encoding 14-3-3γ was also cloned into the NheI/XhoI sites of the pcDNA3.1 vector with an amino-terminal 3Myc tag to produce pcDNA3.1-3Myc-14-3-3γ. cDNAs encoding human Tip60 (GenBank™ accession number 36287048) and AICD (carboxyl-terminal 44 amino acids of human APP695, C44) were amplified by PCR and subcloned into the pcDNA3.1 vector with an amino-terminal EGFP sequence to produce pcDNA3.1-EGFP-Tip60 and pcDNA3.1-EGFP-AICD, respectively. The cDNA encoding APP695 (21Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) was recloned into the NheI/ApaI sites of pcDNA3.1 to generate pcDNA3.1-APP. The cDNA encoding FE65 (24Borg J.P. Yang Y. De Taddeo-Borg M. Margolis B. Turner R.S. J. Biol. Chem. 1998; 273: 14761-14766Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) was recloned into the NheI/NotI sites of pcDNA3.1-N-FLAG to generate pcDNA3.1-FLAG-FE65. cDNA encoding the amino acid sequence between amino acids 313 and 356 of FE65 was deleted by PCR to produce pcDNA3.1-FLAG-FE65Δ1. cDNAs encoding deletion constructs, pcDNA3.1-14-3-3γ-71C (lacking the amino-terminal 70 amino acids of 14-3-3γ) and pcDNA3.1-14-3-3γ-N160 (lacking the carboxyl-terminal 87 amino acids of 14-3-3γ) were prepared by PCR. To construct pcDNA3.1-APP-T668D, substitution of Asp for Thr-668 was performed using site-directed mutagenesis with PCR. pSRα-3HA-JNKK2-JNK1 and pSRα-3HA-JNKK2(K149M)-JNK1 have been described previously (35Engelman J.A. Berg A.H. Lewis R.Y. Lin A. Lisanti M.P. Scherer P.E. J. Biol. Chem. 1999; 274: 35630-35638Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Plasmids encoding proteins fused with green fluorescent protein variants were prepared as follows. cDNA encoding AICD(C44) was cloned into the NheI/XhoI sites of pcDNA3.1-N-CFP to generate pcDNA3.1-CFP-AICD(C44); cDNA encoding human 14-3-3γ was cloned into the NheI/XhoI sites of pcDNA3.1-N-YFP to generate pcDNA3.1-YFP-14-3-3γ; and cDNA encoding human FE65 was cloned into the NheI/NotI sites of pcDNA3.1-N-RFP to generate pcDNA3.1-N-RFP-FE65. cDNA encoding a typical nuclear localization signal (NLS, the NH2-DPKKKRKV-COOH sequence was used) tandemly repeated three times was inserted into the NheI/XhoI sites of pcDNA3.1-N-CFP or pcDNA3.1-N-RFP to generate pcDNA3.1-CFP-NLS or pcDNA3.1-RFP-NLS. Antibodies—Anti-FLAG (M2) mouse monoclonal and anti-APP rabbit polyclonal (αAPP/C) antibodies were purchased from Sigma. Anti-Myc monoclonal antibody was purchased from Invitrogen. Anti-synaptotagmin 1 (SYT) mouse monoclonal antibody (clone 41) was purchased from BD Biosciences. Anti-GST mouse monoclonal antibody was purchased from Upstate Biotechnology, Inc. Anti-EGFP rabbit polyclonal antibody was purchased from Medical and Biological Laboratories Co., Ltd. Anti-14-3-3γ (UT116) rabbit polyclonal antibody was raised against the peptide 14-3-3γ-(235-247)-(Cys), and its specificity was verified (supplemental Fig. 1). An APP phosphorylation state-specific mouse monoclonal antibody, 10A3, was prepared against the Thr-668-phosphorylated AICD peptide (amino acids 649-695 of APP695), and the antibody recognized the phosphorylated peptide but not the nonphosphorylated AICD peptide (supplemental Fig. 2). The anti-APP rabbit polyclonal antibody G369 was supplied by Dr. Gandy, and its specificity has been described (36Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.M. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar). In Vitro Binding Assay—The cDNA encoding 14-3-3γ was recloned into pGEX-4T-1 (Amersham Biosciences) to produce a GST-14-3-3γ fusion protein. The synthetic AICD(C47) peptide (8.3 pmol) with or without phosphate at Thr-668 (21Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) was mixed with 3 pmol of GST-14-3-3γ or 6 pmol of GST alone in HBS-N buffer (10 mm HEPES (pH 7.6), 150 mm NaCl, 0.5% (v/v) Nonidet P-40, 5 μg/ml chymostatin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin A) containing bovine serum albumin (1 mg/ml). The GST fusion protein was recovered by pull down with glutathione beads, and the precipitates were analyzed by Western blotting with the indicated antibodies. Coimmunoprecipitation and Western Blot Analysis—Neuro-2a (N2a) cells (4.5 × 105 cells) were transfected with the indicated plasmids, as described previously (37Sumioka A. Imoto S. Martins R.N. Kitino Y. Suzuki T. Biochem. J. 2003; 374: 261-268Crossref PubMed Scopus (19) Google Scholar). The cells were cultured for 24 h and lysed in HBS-N buffer on ice. After centrifugation (15,000 × g for 10 min), antibody was added to the supernatant of the lysate. The immunoprecipitates were subjected to Western blot analysis using specific antibodies. Brains of wild type mouse (C57BL/6, 8-week-old males) and APP-T668D mutant mouse congenic to C57BL/6 (12-week-old males) were homogenized on ice in a 4-fold volume of buffer (10 mm HEPES (pH 7.4), 0.32 m sucrose, 5 μg/ml chymostatin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin) by 10 strokes of a loose-fitting Dounce homogenizer. The homogenates were centrifuged at 1,000 × g for 7 min, and the supernatants were then further centrifuged at 2,000 × g for 30 min. The resulting precipitates were lysed for 1 h on ice in HBS-N buffer. After centrifugation at 10,000 × g for 1 h at 4 °C, 0.3 μl of UT116 antiserum or 1 μg of nonimmune rabbit IgG was added to the supernatant of the HBS-N lysate together with protein G-Sepharose. The immunoprecipitate was eluted by addition of the UT116 antigen peptide (1.5 mg/ml), and the eluate was analyzed by Western blotting with specific antibodies. Gene Transactivation Assays—The cDNA of APP695 was inserted into the NheI/NotI sites of pBIND-C-Gal4BD to construct pBIND-APP-Gal4BD, which has a Gal4 DNA-binding domain inserted into the 3′ end of APP. To construct pBIND-AICD(C44)-Gal4BD, pBIND-C30-Gal4BD, pBIND-C20-Gal4BD, pBIND-C49-Gal4BD, and pBIND-AICD-(T668D)-Gal4BD, the cDNAs that encode the respective amino acids at positions 652-695, 666-695, and 676-695 of APP695 were generated by PCR using pcDNA3.1-APP or pcDNA3.1-APP-T668D as templates and cloned into the NheI/XhoI sites of pBIND-C-Gal4BD. To construct pBIND-S-rich-C20-Gal4BD, the cDNA that encodes amino acids 191-220 of XB51α (37Sumioka A. Imoto S. Martins R.N. Kitino Y. Suzuki T. Biochem. J. 2003; 374: 261-268Crossref PubMed Scopus (19) Google Scholar) was generated by PCR using pcDNA3.1-FLAG-XB51α as a template and inserted into the 5′ end of pBIND-C20-Gal4BD. The cDNA of Tip60 was recloned into the NheI/XhoI sites of pBIND-N-Gal4BD to construct pBIND-Gal4BD-Tip60, which had a Gal4 DNA-binding domain inserted into the 5′ end of Tip60. N2a cells (1.8 × 104 cells) plated in 96-multiwell plates were transiently transfected, using LipofectAMINE2000 (Invitrogen), with the indicated amounts of pBIND plasmids and pG5luc (Promega), a reporter luciferase gene having a Gal4 DNA-binding sequence on its promotor, in the presence of various cDNAs inserted into the pcDNA3.1 vector. To standardize the overall amount of plasmid used, empty vector was added. The transcriptional activity of the reporter gene was analyzed by using the dual luciferase assay system (Promega). All of the combinations were tested in quadruplicate, and the luciferase activity was normalized according to the manufacturer's protocol to eliminate the effect of transfection efficiency differences. Knock down Study with RNAi—cDNAs of small interfering RNA were designed against the human 14-3-3γ (nucleotides 635-654) or 14-3-3ϵ (nucleotides 282-300) genes and subcloned into the pSuper vector (OligoEngine). N2a cells (1.2 × 104 cells) were transfected with 200 ng of pSuper14-3-3γ or pSuper14-3-3ϵ in Lipofectamine 2000, and the expression level of endogenous 14-3-3γ was examined by Western blot analysis with the UT116 antibody. Intracellular Localization of Fluorescent Protein Fusion Proteins—N2a cells were transfected with the indicated plasmids encoding GFP fusion proteins using Lipofectamine 2000 (Invitrogen). The cells were cultured for 24 h and viewed using a confocal laser scanning microscope (LSM510, Carl Zeiss). Association of 14-3-3γ with Nonphosphorylated APP and AICD—Comprehensive screening of a human brain cDNA library with the yeast two-hybrid system using the cytoplasmic domain of APP as bait resulted in isolation of cDNA clones encoding a part of the 14-3-3γ protein (data not shown). Thus, we prepared complete cDNA clones encoding 14-3-3 isoforms evolutionarily close to 14-3-3γ, except for 14-3-3δ (Fig. 1a), and we examined their association with APP by coimmunoprecipitation assays (Fig. 1b). N2a cells expressing amino-terminal FLAG-tagged 14-3-3β, -ϵ, -γ, -η, or -θ isoforms and APP were lysed and subjected to coimmunoprecipitation assays with anti-FLAG antibody. The immunoprecipitates (IP) and cell lysates (lysate) were analyzed by Western blotting with anti-FLAG and anti-APP antibodies. APP was recovered in FLAG-14-3-3γ and FLAG-14-3-3η immunoprecipitates, but 14-3-3β, 14-3-3ϵ, and 14-3-3θ failed to coimmunoprecipitate APP. Control antibody from nonimmunized animals did not recover either 14-3-3γ or APP, indicating that the intracellular interaction between 14-3-3γ and APP is specific (Fig. 1c). Because FLAG-14-3-3γ binding to APP was stronger than that of FLAG-14-3-3η (Fig. 1b) and both 14-3-3γ and 14-3-3ϵ have been associated with AD (38Fountoulakis M. Cairns N. Lubec G. J. Neural Transm. 1999; 57: 323-335Google Scholar), we focused our analysis on the role of 14-3-3γ in APP function. Among the postulated APP functions, such as providing a cargo receptor on transport vesicles (27Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 39Kamal A. Stokin G.B. Yang Z. Xia C.-H. 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Thus, we analyzed the interaction of AICD with 14-3-3γ. A GST-14-3-3γ fusion protein was incubated with a synthetic AICD peptide (649-695 of APP695, C47), with or without phosphorylation of the Thr-668 residue, which is a major phosphorylation site in brain and cultured cells (41Iijima K. Ando K. Takeda S. Satoh Y. Seki T. Itohara S. Greengard P. Kirino Y. Nairn A.C. Suzuki T. J. Neurochem. 2000; 75: 1085-1091Crossref PubMed Scopus (206) Google Scholar, 42Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 43Taru H. Suzuki T. J. Biol. Chem. 2004; 279: 21628-21636Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Many 14-3-3 proteins bind to phosphorylated target proteins and regulate their function and/or structure (44van Hemert M.J. Steensma H.Y. van Heusden G.P. BioEssays. 2001; 23: 936-946Crossref PubMed Scopus (472) Google Scholar). The GST-14-3-3γ, however, bound the nonphosphorylated AICD peptide but did not bind AICD peptide phosphorylated at Thr-668 (Fig. 1d). The physiological significance of this interaction was confirmed using mouse brain samples (Fig. 1e). The 14-3-3γ-specific antibody UT116 (supplemental Fig. 1) was used to immunoprecipitate the protein from mouse brain lysates, and the immunoprecipitate was analyzed by Western blotting for 14-3-3γ, APP, APP phosphorylated at Thr-668, and synaptotagmin 1. The 14-3-3γ antibody coimmunoprecipitated APP but not Thr-668-phosphorylated APP and synaptotagmin 1, indicating that the association of 14-3-3γ with APP occurs in brain and the phosphorylation of APP interferes with the interaction (Fig. 1e, left). Because we have established a mutant mouse line, in which the phosphorylation site Thr-668 in APP is altered to Asp-668 and Ala-668, 4K. Seki, S. Takeda, Y. Sano, T. Nakaya, E. Kawaguchi, T. Suzuki, and S. Itohara, submitted for publication. we examined the association of 14-3-3γ with APP in the T668D mutant mouse brain. 14-3-3γ was immunoprecipitated from a mutant mouse brain lysate using the 14-3-3γ-specific antibody UT116, and the immunoprecipitate did not contain the T668D APP (Fig. 1e, right). AICD was not detected in these assays, and this may be due to the small amounts or very rapid catabolism of AICD in brain. We have reported previously that phosphorylation of Thr-668 in the 667VTPEER672 motif suppressed the interaction of AICD with FE65, which recognizes the 681GYENPTY687 motif, because the phosphorylation of Thr-668 affects the amino-terminal helix capping-box structure composed of 667VTPEER672 and the helical state of the following amino acid sequence that includes the 681GYENTPY687 FE65-binding motif (21Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 22Ramelot T.A. Gentile L.N. Nicholson L.K. Biochemistry. 2000; 39: 2714-2725Crossref PubMed Scopus (76) Google Scholar, 23Ramelot T.A. Nicholson L.K. J. Mol. Biol. 2001; 307: 871-884Crossref PubMed Scopus (129) Google Scholar). To confirm that 14-3-3γ binds to APP through the 667VTPEER672 motif, we examined 14-3-3γ binding to other motifs. 14-3-3γ failed to bind to APP when a region containing the 667VTPEER672 motif was deleted, but not when regions containing the 653YTSI656 or 681GYENPTY687 motifs were deleted (supplemental Fig. 3). Therefore, our observations confirm that 14-3-3γ binds to the 667VTPEER672 motif, that Thr-668 in this motif is important for the 14-3-3γ-APP interaction, and that the phosphorylation of Thr-668 suppresses the association of APP and AICD with 14-3-3γ. Association of 14-3-3γ with AICD Facilitates FE65-dependent Gene Transactivation—Because 14-3-3γ associated with AICD, we explored the effect of 14-3-3γ on FE65-dependent gene transactivation mediated by AICD. pBIND-AICD(C44)-Gal4BD and pG5luc were transfected in N2a cells expressing different isoforms of 14-3-3, in the presence (+) or absence (-) of FE65. The AICD-Gal4BD fusion protein showed FE65-dependent gene transactivation (Fig. 2a, compare lane 2 with lane 1), as reported previously (11Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1054) Google Scholar). Coexpression of 14-3-3γ or 14-3-3η enhanced this activity to 2-2.5-fold, respectively (Fig. 2a, compare lanes 5 and 6 with lane 2), which is consistent with the affinity of their binding to APP (Fig. 1b). AICD(C49), a product of the ϵ-site cleavage of APP (45Gu Y. Misonou M. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), is the form predominantly observed in cells (46Sato T. Dohmae N. Qi Y. Kakuda N. Misonou H. Mitsumori R. Maruyama H. Koo E.D. Haass C. Takio K. Morishima-Kawashima M. Ishiura S. Ihara Y. J. Biol. Chem. 2003; 278: 24294-24301Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Similar to AICD(C44) (Fig. 1b and Fig. 2a), AICD(C49) mediated FE65-dependent gene transactivation, and transactivation was enhanced by 14-3-3γ (supplemental Fig. 4). Transactivation by other 14-3-3 isoforms was absent or minimal (Fig. 2a, compare lanes 3, 4, 7, and 8 with lane 2). The e