Role of 14-3-3γ in FE65-dependent Gene Transactivation Mediated by the Amyloid β-Protein Precursor Cytoplasmic Fragment*

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.

cated at the ␣ and ␤ sites, respectively) is secreted, and then following the second cleavage, p3 or ␤-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 (3,4). 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 ␥-secretase (5)(6)(7)(8). Cleavage of these type I membrane proteins generates and releases their cytoplasmic domain, which is thought to play an important role, together with other transcriptional regulatory factors, in gene transactivation (9,10).
The cytoplasmic domain fragment derived from APP (AICD) activates the transcription of a reporter gene in the presence of the neuronspecific adaptor protein FE65 (11,12) 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 (13)(14)(15)(16)(17)(18)(19). The AICD contains several functionally important motifs. The 653 YTSI 656 motif (human APP695 isoform numbering) is the basolateral sorting signal of APP in Madin-Darby canine kidney epithelial cells (20). The 667 VTPEER 672 motif contains the phosphorylation site Thr-668 and controls the stability of the overall structure of AICD (21)(22)(23). The 681 GYENPTY 687 motif regulates metabolism and transport of APP through interactions with various adaptor proteins such as X11s, FE65s, JIPs, and AIDA-1 (24 -31). Thus, the 681 GYENPTY 687 motif is thought to be essential for AICD function in FE65-dependent gene transactivation. However, it remains uncertain whether other motifs regulate AICD function and whether other protein factors play an important role in the regulation of AICDmediated FE65-dependent gene transactivation. 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 667 VTPEER 672 motif regulates AICD function in FE65-dependent gene transactivation through the phosphorylation of Thr-668 and interactions with the 14-3-3␥ protein.
Mammalian 14-3-3 proteins are a ubiquitously expressed gene family with seven distinct isoforms that are involved in various signal transduction pathways (32). 14-3-3␥ is one of the most abundant isoforms in brain, skeletal muscle, and heart (33,34). We found that association of the 14-3-3␥ dimer with both nonphosphorylated AICD and FE65 facilitates gene expression, whereas the phosphorylation of AICD interferes with 14-3-3␥ 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 (21), suggest that the AICD 667 VTPEER 672 motif is an important regulatory region for AICD-mediated FE65-dependent gene transactivation.
Coimmunoprecipitation and Western Blot Analysis-Neuro-2a (N2a) cells (4.5 ϫ 10 5 cells) were transfected with the indicated plasmids, as described previously (37). The cells were cultured for 24 h and lysed in HBS-N buffer on ice. After centrifugation (15,000 ϫ 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 g/ml chymostatin, 5 g/ml leupeptin, and 5 g/ml pepstatin) by 10 strokes of a loose-fitting Dounce homogenizer. The homogenates were centrifuged at 1,000 ϫ g for 7 min, and the supernatants were then further centrifuged at 2,000 ϫ g for 30 min. The resulting precipitates were lysed for 1 h on ice in HBS-N buffer. After centrifugation at 10,000 ϫ g for 1 h at 4°C, 0.3 l of UT116 antiserum or 1 g 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.
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.
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).
Moreover, we have confirmed that C20 and S-rich-C20 bind to FE65, but these interactions are weak compared with C30 binding (data not shown). These studies clearly showed that association of 14-3-3␥ with AICD facilitated FE65-dependent gene transactivation. These observations supported our hypothesis that AICD-mediated FE65-dependent gene transactivation requires binding of 14-3-3␥ to the 667 VTPEER 672 motif to express higher activity.
Phosphorylation of Thr-668 in the 667 VTPEER 672 motif blocked the interaction of AICD and APP with 14-3-3␥ (Fig. 1, d and e). Thus, we examined the relationship between Thr-668 and FE65-dependent gene transactivation activity. When lysates of N2a cells expressing both FLAG-14-3-3␥ and the wild type or T668D mutant of APP, in which Thr-668 is altered to Asp668, were analyzed by coimmunoprecipitation, the anti-FLAG antibody failed to recover the T668D mutant APP (Fig.  3c). The T668D AICD-Gal4BD also lost the activity to facilitate FE65dependent gene transactivation by 14-3-3␥ (Fig. 3d, right columns). In this study, the T668D mutation decreased its activity slightly in the 5 A. Sumioka, unpublished observations. absence of 14-3-3␥ (Fig. 3d, middle columns) because the mutation at the Thr-668 residue causes a conformational change in the carboxylterminal region containing the FE65-binding motif (21). We induced the phosphorylation of AICD in cells by expressing a JNKK2-JNK1 fusion protein, which is a constitutively active Jun kinase (Fig. 3e) (35). Because recent findings reported that JNK could phosphorylate APP at Thr-668 in cultured cells under stress conditions (43,47), the expression of the JNKK2-JNK1 fusion protein is expected to increase the phosphorylation of AICD at Thr-668 in cells. When AICD-EGFP was expressed in N2a cells with wild type JNKK2-JNK1 (WT), phosphorylated AICD-EGFP (pAICD-EGFP) appeared regardless of the coexpression of FE65 and 14-3-3␥ (Fig. 3e). When a mutant JNKK2-JNK1 (KM) which carries a Lys149Met mutation on JNKK2 and has no kinase activity (35) was coexpressed, no phosphorylation of AICD-EGFP was observed. Using this system the effect of AICD phosphorylation on FE65dependent gene transactivation was measured (Fig. 3f). In the presence of mutant JNKK2-JNK1 (KM), 14-3-3␥ facilitated AICD-mediated FE65-dependent gene transactivation. However, the expression of wild type JNKK2-JNK1 blocked this effect (Fig. 3f, right columns). These observations indicate that the phosphorylation of Thr-668 in the 667 VTPEER 672 motif inhibits the 14-3-3␥ enhancement of AICD-mediated FE65-dependent gene transactivation, which is due to dissociation of 14-3-3␥ from AICD.
We further investigated whether deletion of FE65 amino acids 313-356 affects association with Tip60 (Fig. 5d), because FE65-Tip60 interactions have been reported previously (11). FLAG-FE65 or FLAG-FE65⌬1 was expressed in cells with EGFP-Tip60, and lysates were coimmunoprecipitated with the anti-FLAG antibody. Western blot analysis to detect immunoprecipitates revealed that Tip60 bound both wild type (WT) FE65 and FE65⌬1, indicating the FE65⌬1 retains the ability to bind Tip60.

DISCUSSION
The 14-3-3 protein family is known to play an important role in many intracellular processes, including apoptosis, the cell cycle, and signal transduction (32,44). Interaction of target proteins with 14-3-3 has been useful in characterizing the molecular mechanisms of these cellular events, because 14-3-3 proteins are expressed abundantly in various tissues and provide scaffolding for many intracellular reactions. In this report, we demonstrated that interactions between AICD and 14-3-3␥ are regulated by phosphorylation of AICD at Thr-668. We proposed that the 14-3-3␥ protein contributes AICD-mediated FE65-dependent gene transactivation (Fig. 7). As a first step, AICD associates with FE65, and the dimeric 14-3-3␥ binds to these proteins to form an active complex composed of AICD, FE65, and 14-3-3␥, which mediates FE65-dependent gene transactivation. When AICD was phosphorylated at Thr-668, 14-3-3␥ fails to associate with the initial complex composed of AICD and Fe65, because phosphorylated AICD cannot bind to 14-3-3␥, or suppresses the association with FE65 (21). Thus, the phosphorylation of AICD at Thr-668 results in failure to form the active ternary complex composed of AICD, FE65, and 14-3-3␥ and down-regulates AICD-mediated FE65-dependent gene transactivation. Therefore, the phosphorylation of AICD at Thr-668 may act as a molecular switch for FE65-dependent gene transactivation mediated by AICD. The complex may be initially formed in the cytoplasm and translocated into the nucleus, because all three proteins colocalized in both the cytoplasm and nucleus when coexpressed.
Several recent studies have reported that AICD shows transactivation activity in the presence of FE65 in cellular models, but the mechanism of AICD modulation of FE65-dependent gene transactivation remains unclear. During the preparation of this manuscript, Cao and Südhof (15) reported that gene transactivation mediated by Tip60 depends on the APP holoprotein but is independent of AICD. However, in our study, using a construct in which the PI1 domain of FE65 is deleted, AICDmediated FE65-dependent gene transactivation was observed despite the lack of interaction between Fe65 and Tip60. Furthermore, in the gene transactivation assays in our study, using the FE65⌬1 construct, which is not able to interact with 14-3-3␥, AICD-mediated FE65-dependent gene transactivation was almost completely lost. Taken together, these data suggest that AICD-mediated gene transactivation depends on 14-3-3␥ rather than Tip60. Therefore, new models of the mechanism of FE65-dependent gene transactivation may be needed. We demonstrated that expression of 14-3-3␥ enhanced the interaction between AICD and FE65, and the down-regulation of endogenous 14-3-3␥ expression by RNAi suppressed this interaction. 14-3-3␥ expression was correlated with the facilitation of gene transactivation activity, and the lack of 14-3-3␥ was correlated with suppression of transactivation. The molecular mechanism by which these regulated interactions controlled gene transactivation will be the subject of future studies. Further analyses will be needed to reveal the molecular mech-anisms of nuclear translocation of FE65 and of gene transactivation mediated by the interaction of AICD with 14-3-3␥.
14-3-3 family proteins interact with various proteins in a phosphorylation-dependent manner and provide scaffoldings for molecules mediating cellular events (32,44). Among several motifs in AICD, the 667 VTPEER 672 motif contains Thr-668, which is phosphorylated (26, 36, 40 -42). In a previous study, we reported that the constitutive phosphorylation of APP at Thr-668 was observed specifically in brain (41). The phosphorylation of APP at Thr-668 is mediated by neuronal cyclin-dependent protein kinase 5 (41), stress-activated protein kinase (43,45), or glycogen synthase kinase 3␤ (49). Neuron-specific phosphorylation of APP at Thr-668 is thought to be important for neuronal functions of APP, because the phosphorylation of APP is correlated with the extension of neurites in differentiating PC12 cells (40,50). We found that nonphosphorylated APP and AICD interact with 14-3-3␥, although reports of interactions between 14-3-3 and nonphosphorylated proteins are few. We found that the interaction of APP and AICD with 14-3-3␥ is prevented by the phosphorylation of APP at Thr-668, both in vivo and in vitro. Furthermore, we reported previously that the phosphorylation of APP suppresses FE65 binding to the 681 GYENPTY 687 motif of AICD by altering the AICD conformation (21). Thus, the phosphorylation of AICD at Thr-668 comprehensively regulates AICD-mediated FE65-dependent gene transactivation through the dissociation of FE65 and 14-3-3␥ from AICD. Further investigation into the function of AICD, FE65, and 14-3-3␥ in AICD-mediated Fe65-dependent gene transactivation will contribute to characterization of APP function and may help to understand the neuronal degeneration process in AD.