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Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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APP is a phosphoprotein and predominantly phosphorylated at the Thr668 residue in brain (the residue numbering follows that used for the APP695 isoform) (Iijima et al. 2000). Thr668 is located in the 667-VTPEER-672 motif, which contains an amino-terminal helix capping-box structure that helps to stabilize the neighbor-ing carboxyl-terminal helix structure (Ramelot et al. 2000). Several protein kinases, such as cyclin-dependent protein kinase 5 (Cdk5) (Iijima et al. 2000), p34cdc2 protein kinase (cdc2) (Oishi et al. 1997), glycogen synthase kinase 3ß (GSK-3ß) (Aplin et al. 1996) and c-jun N-terminal kinases (JNK) (Standen et al. 2001; Taru et al. 2002; Kimberly et al. 2005) are responsible for phosphorylating Thr668 in several cell types under various culture conditions. This phosphorylation correlates with neurite outgrowth in PC12 cells (Ando et al. 1999), regulates the interaction of APP and AICD with an adaptor protein FE65 (Ando et al. 2001), affects the stabilization of AICD by FE65 (Kimberly et al. 2005) and regulates hippocampal plasticity (K. Seki, S. Takeda, Y. Sano, E. Kawaguchi, T. Nakaya, T. Suzuki and S. Itohara, unpublished observations). However, the detailed molecular mechanisms of APP phosphorylation in these physiological events remain unknown. Because the phosphorylation of Thr668 results in destabilization of the amino-terminal helix capping-box structure (Ramelot & Nicholson 2001) and leads to an overall conformational change in the cytoplasmic domain including the FE65-binding motif 681-GYENPTY-687 (Ando et al. 2001), an aberrant phosphorylation may disable the conformational switch and may affect the function of APP and/or interfere with AICD-mediated FE65-dependent events.
The neuron-specific adaptor protein FE65 interacts with the cytoplasmic domain of APP in a tyrosine phosphorylation-independent manner through its second phosphotyrosine interaction (PI) domain (Fiore et al. 1995; Borg et al. 1996). Although this interaction is known to modulate APP metabolism including the generation of Aß (Sabo et al. 1999; Ando et al. 2001), a recent study has suggested another role where the complex composed of AICD and FE65 may transactivate genes after translocating into the nucleus (Cao & Sudhof 2001). It was also shown that the APP holoprotein can anchor FE65 in the cytoplasmic membrane and prevent the translocation of FE65 into the nucleus (Sabo et al. 1999, 2003; Minopoli et al. 2001; Scheinfeld et al. 2002). However, it is still unclear how the translocation of FE65 and AICD into the nucleus from the cytoplasm and/or membrane is regulated and events in the nucleus, such as gene transactivation, are accomplished, although a possible mechanism has been proposed whereby AICD is generated by 
-secretase cleavage as a complex with FE65 (Cao & Sudhof 2004).
In this study, we analyzed the role of the Thr668 residue in regulating the translocation of FE65 and AICD into the nucleus and the FE65-dependent gene transactivation mediated by AICD. We found that the translocation of AICD into the nucleus is not regulated by the phosphorylation of AICD nor by the presence of FE65 or its association with AICD. However, the FE65 tethered to the membrane by the APP holoprotein was liberated from membrane by phosphorylation or amino acid substitution of APP at Thr668, which up-regulated gene transactivation mediated by AICD. Thus, phosphorylation of APP holoprotein but not AICD may serve as a molecular switch of AICD-mediated FE65-dependent gene transactivation by regulating the translocation of FE65.
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Results |
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Previously we reported that the APP695 holoprotein is phosphorylated at Thr668 in neuronal tissues (Iijima et al. 2000), and this phosphorylation suppresses the interaction of APP and its cytoplasmic peptide with FE65 by causing an overall conformational change in the cytoplasmic domain that contains the FE65-binding motif (Ando et al. 2001). However, phosphorylation at Thr668 of AICD in brain has not been analyzed. Therefore, we first examined the phosphorylation state of AICD in vivo. Immunoblotting of mouse brain proteins with an anti-APP carboxyl-terminal domain antibody detected two protein bands smaller than the 6.5-kDa protein size standard (lane 1 of Fig. 1A), and the intensity of both bands increased following incubation of membrane fraction (data not shown), which produces AICD in vitro (Gu et al. 2001). When the brain lysate was treated with 
PPase, only the faster migrating protein was observed, and the slower migrating protein disappeared (lane 2 of Fig. 1A). The anti-phospho Thr668 antibody recognized only the slower migrating protein (lane 3 of Fig. 1A), and this band disappeared after the phosphatase treatment (lane 4 of Fig. 1A). These observations indicate that the slower migrating protein is the AICD phosphorylated at Thr668 (pAICD), and the faster migrating protein represents AICD that is not phosphorylated (nAICD), at least on the Thr668 residue.
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(Araki et al. 2003), amyloid precursor-like protein 2 (APLP2) (Scheinfeld et al. 2002), calnexin, presenilin NTF (Zhang et al. 1998) and APP CTFs were recovered in the membrane fraction and -tubulin was recovered in the cytosolic fraction (Fig. S1), as expected. No membrane or cytosolic proteins were recovered in the nuclear fraction (Figs 1B and S1). Therefore, we judged that the subcellular fractionation was complete. APP phosphorylated at Thr668 was found only in the wild-type mouse, and no APP phosphorylated at Thr668 was observed in the mutant mouse (fourth panel in Fig. 1B). In this experiment, large amounts of FE65 were detected in the membrane and cytosolic fractions and little was found in the nuclear fraction (second panel in Fig. 1B). Overall, there was no difference in FE65 distribution between the wild-type and mutant mouse, except that membrane FE65 was present in slightly lower amounts in the mutant mouse (see Fig. 3E). Both pAICD and nAICD were detected in the membrane fraction of the wild-type mouse and AICDA was observed in the membrane fraction of the mutant mouse (top panel in Fig. 1B). In the nuclear fraction of the wild-type mouse, nAICD was dominantly detected and an extremely little pAICD was found. In the mutant mouse, AICDA was detected in the nuclear fraction, although it has been reported that both the Thr substitution for Ala and the phosphorylation of Thr suppressed interaction with FE65 (Ando et al. 2001). These observations suggest that phosphorylation of AICD and association of FE65 are not essential for the nuclear translocation of AICD in vivo.
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AICD has been reported to mediate FE65-dependent gene transactivation (Cao & Sudhof 2001; Baek et al. 2002; Telese et al. 2005; Pardossi-Piquard et al. 2005), although the mechanism has not been clearly elucidated (Cao & Sudhof 2004; Hass & Yankner 2005). If AICD is constitutively translocated into the nucleus by a mechanism that is not coupled to the nuclear translocation of FE65, the regulation of the nuclear translocation of FE65 itself may be a key event in AICD-mediated FE65-dependent gene transactivation. FE65 is anchored to the membrane by its association with the APP holoprotein when these two proteins are co-expressed in the cell (Minopoli et al. 2001; Sabo et al. 2003). Thus, the dissociation of FE65 from membrane-associated APP could be a trigger for AICD-mediated cellular signaling. We therefore first examined an AICD-mediated FE65-dependent gene transactivation assay in the presence of membrane-associated APP. When Gal4BD-AICD was co-expressed with FE65, gene transactivation activity was detected (compare column 2 with column 1 in Fig. 3B), and this activity decreased in the presence of APP695 (compare column 2 with column 3). When Gal4BD-AICD was coexpressed without FE65, the activity was weakly increased (approximately threefold), which is a comparable level with column 3 (data not shown), suggesting that the membrane-associated APP knocked down the FE65-dependent gene transactivation activity mediated by AICD. This result suggests two possibilities. One is that APP holoprotein tethered FE65 to the membrane and FE65-dependent gene transactivation activity mediated by AICD was suppressed. Another is that AICD derived from exogenous APP competes with Gal4BD-AICD for FE65-binding in the nucleus. The second possibility can be excluded because a -secretase inhibitor (L-685, 458), which suppresses the generation of AICD from APP holoprotein, had no effect (compare columns 2 and 3 with columns 5 and 6). The inhibition of -secretase activity was confirmed in a study using APPNGal4BD-AICD, in which Gal4BD was inserted into a region of APP toward the carboxyl-terminus of -cleavage site. APPNGal4BD-AICD did not generate Gal4BD-AICD in the presence of L-685, 458 and show the gene transactivation activity (data not shown). These results strongly suggest that FE65 can constitutively bind to the APP holoprotein and this association negatively regulates the AICD-mediated gene transactivation.
It has already been reported that the interaction of APP and AICD with FE65 is suppressed by the phosphorylation of APP and AICD at Thr668 (Ando et al. 2001). Therefore, FE65 anchored to the membrane by its association with APP may be released into the cytoplasm by phosphorylation of APP. Then the liberated FE65 translocates into the nucleus, where it associates with nAICD that has already moved there by an FE65-independent mechanism. To evaluate this hypothesis, we performed an FE65-dependent gene transactivation assay using the wild-type and mutant APP695 constructs; NFLAG-APPwt, NFLAG-APPT668A and NFLAG-APPT668E (Fig. 3C). Expression of NFLAG-APPwt suppressed FE65-dependent gene transactivation activity. Expression of NFLAG-APPT668A and NFLAG-APPT668E restored the FE65-dependent gene transactivation activity, because both amino acid substitutions suppress the interaction of FE65 with APP as well as phosphorylation at Thr668 (Ando et al. 2001).
Membrane anchoring of FE65 by APP was examined in the brains of mutant mice expressing APPT668A (Fig. 3D). Wild (WT) and mutant mouse brains were subjected to fractionation and proteins in the membrane and cytosolic fractions were compared by Western blot analysis. No remarkable quantitative differences were observed in the membrane and cytosolic proteins between the wild-type and mutant mice (Fig. 1B). However, membrane-anchored FE65 decreased to approximately 75% in the mutant mouse relative to that in the wild-type mouse brain, whereas the amounts of APP and cytosolic FE65 were identical in the wild-type and mutant mouse brains (Fig. 3D,E). These data suggest that Thr668 regulates the anchoring of FE65 to APP through its phosphorylation state. However, FE65 may interact with not only APP but also other membrane proteins (Trommsdorff et al. 1998; Kinoshita et al. 2001), allowing much of it to remain associated with the membranes.
The anchoring of FE65 to APP and nuclear localization of FE65 were demonstrated by testing the cellular localization of FE65 in the presence of the APP holoprotein (Fig. 4). The majority of NHA-FE65 was detected in the cytoplasm and a minority in the nucleus (Fig. 4A,a-c). In this study, we used Gal4BD-AICD instead of AICD, because Gal4BD-AICD spontaneously translocates into the nucleus due to the nuclear localization signal of Gal4BD. In contrast to this, AICD could not be detected with immunostaining in nucleus because of its small amount (data not shown). Gal4BD-AICD may mimic the endogenous AICD at the point of its constitutive translocation into the nucleus in a FE65-independent manner. Thus, the Gal4BD-AICD and Gal4BD proteins largely localized in the nucleus (Fig. 4A,d-i). When NHA-FE65 was co-expressed in cells with Gal4BD, the localization of NHA-FE65 was not affected (Fig. 4B,a-d). When NHA-FE65 was expressed in the presence of Gal4BD-AICD, the majority of NHA-FE65 was localized in the nucleus with Gal4BD-AICD (Fig. 4B,e-h). This suggests that FE65 constitutively moves into the nucleus in small amounts using its own nuclear localization signal and then leaves the nucleus. In the presence of Gal4BD-AICD, FE65 is likely to be trapped in the nucleus.
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To examine the effects of APP Thr668 phosphorylation on FE65 localization, APP phosphorylation was induced by ectopic expression of JNK11 in cells under stress conditions (Taru & Suzuki 2004). Cells expressing the indicated combination of APP695, NFLAG-FE65 and HA-JNK11 were treated with or without 0.5 M sorbitol for 45 min. The cells were then subjected to fractionation into membrane and cytosolic fractions, and proteins in the respective fractions were detected by Western blotting. Phosphorylation of APP increased in the membrane fraction of cells with osmotic stress (compare lane 9 with lanes 3 and 6 in the second panel of Fig. 5A, and the results are quantified in Fig. 5B). NFLAG-FE65 was largely recovered in the cytosolic fraction (lane 10–18 in top panel of Fig. 5A), but it was detected in the membrane fraction when APP was expressed (compare lane 3 with lane 2 in top panel), indicating that the expression of APP tethered NFLAG-FE65 to the membrane, as seen in Fig. 4. When the phosphorylation of APP was increased, the amount of tethered NFLAG-FE65 significantly decreased (compare lanes 6 and 9 with lane 3 in the top and second panels in Fig. 5A, and the results were quantified in Fig. 5C). Surprisingly, a small amount of NFLAG-FE65 was tethered to membrane after stimulation of cells with 0.5 M sorbitol, even in the absence of APP (compare lane 8 with lane 9 in the top panel of Fig. 5A), suggesting that a second mechanism may tether a restricted amount of NFLAG-FE65 to the membrane after stimulation with 0.5 M sorbitol. However, our observations clearly indicate that FE65 was tethered to membrane through APP binding and the tethered FE65 was released from APP by phosphorylation at Thr668.
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Discussion |
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It seems to be contradictory that AICD generated by -secretase cleavage is attached to the membrane in spite of its cytoplasmic release. However, the majority of endogenous AICD in brain (Fig. 1B) and cells (Fig. 2A) were recovered in the membrane fraction, and we detected a small amount of exogenous AICD in the membrane fraction when FE65 was co-expressed (Fig. 2A). Therefore, a very small percentage of exogenously expressed AICD may exhibit the physiological behavior similar to endogenous AICD.
The in vitro-secretase assay, in which AICD is generated by incubating the membrane fraction (Gu et al. 2001), showed that the majority of AICD was released into the soluble fraction in spite of the association with the membrane. In contrast, little AICD was recovered in the cytosolic fraction in vivo. Together with these pioneer observations, it was reasonable to lead to the hypothesis that AICD was released into cytoplasm after its generation and degraded rapidly. It had been believed that AICD is subjected to stabilization by binding with FE65 in cytoplasm and the complex was transported into the nucleus. This hypothesis now seems to be reconsidered, because exogenously expressed FE65 did not stabilize endogenous AICD and, moreover, the majority of exogenously expressed AICD did not localize in the nucleus even in the presence of FE65 (Fig. 2A). It is still not obvious how brain AICD becomes localized in the nucleus when only a small amount of AICD exists in the cytoplasm. However, we detected AICDA as well as nAICD in the nucleus in vivo. This observation supports the idea that FE65 cannot accompany AICD into the nucleus, because the binding of FE65 is strongly suppressed by amino acid substitution at Thr668 (Ando et al. 2001), and indicates that the phosphorylation of AICD at Thr668 is not related to its nuclear localization.
The second finding is that APP phosphorylation regulates FE65-dependent gene transactivation mediated by AICD. Phosphorylation of APP at Thr668 clearly decreased the amount of membrane-associated FE65 (Fig. 5), which was also decreased in the mutant mouse brain expressing APPT668A. These findings support the idea that APP phosphorylation is a molecular switch that controls the intracellular distribution of FE65. However, the cytoplasmic region of several other proteins, such as APLPs and low-density lipoprotein receptor-related protein, can also bind FE65 in cells (Trommsdorff et al. 1998; Kinoshita et al. 2001). Thus, APP may not be the only molecular switch controlling FE65 distribution. However, we have demonstrated that APLP2 is phosphorylated in an identical manner to APP (Suzuki et al. 1997; Taru & Suzuki 2004) and the phosphorylation of APLP2 suppresses its interaction with FE65 (Ando et al. 2001). Thus, we can state that, at least, APP and APLP2 can function as molecular switches to control the intracellular distribution of FE65 and, thus, the phosphorylation of APP and/or APLP2 can regulate FE65-dependent gene transactivation mediated by AICD.
Questions may arise as to how FE65 released from APP-anchoring can be distinguished from FE65 that has already been reserved in cytoplasm and translocated into nucleus selectively. A possible interpretation is that interaction of FE65 with membrane-anchored APP induces the conformational change or modification of FE65 molecule. This idea was proposed in previous report (Cao & Sudhof 2004), although the report did not describe the substantial analysis of FE65 and mentioned that FE65 is liberated from membrane by -cleavage of APP as a complex including AICD, which differs from our conclusion. A more probable interpretation is that the binding of APP to the second PI domain of FE65 may open other binding sites such as the WW and the first PI domains by inducing conformational change of FE65. This may facilitate the association of FE65 with unidentified protein factor(s) on the scaffolding of APP, and FE65 may release from APP by APP phosphorylation as a complex with unidentified protein(s) but not with AICD, which may be needed for nuclear transportation of FE65.
APP Thr668 is constitutively phosphorylated in neuronal tissues (Iijima et al. 2000) and a stress stimulus can induce this phosphorylation even in non-neuronal cells (Taru & Suzuki 2004). Several physiological functions have been proposed for APP phosphorylation. Phosphorylated APP is largely localized in the neurite tips of neuronal cells (Ando et al. 1999; Iijima et al. 2000; Muresan & Muresan 2004), and neurite tips or growth cones play an important role in axonal guidance and in receiving surrounding information (Henley & Poo 2004). APP may therefore function to transmit extracellular stimuli into the nucleus through FE65 to cause the gene expression, which may be regulated by the association of AICD with FE65 and the phosphorylation of membrane APP. Therefore, it will be important to identify more of the target gene(s) whose expression is regulated by FE65 and AICD or the phosphorylation state of APP, although several genes, including KAI1 and neprilysin, have been reported as target genes (Baek et al. 2002; Telese et al. 2005; Pardossi-Piquard et al. 2005). The present findings that AICD translocation into the nucleus is not dependent on AICD phosphorylation and FE65-dependent gene transactivation mediated by AICD in the nucleus are regulated by APP phosphorylation may provide a starting point for understanding AICD function in AD pathobiology.
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Experimental procedures |
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The polyclonal anti-APP rabbit antibody G369 has been previously characterized (Oishi et al. 1997). The polyclonal anti-FE65 antibody UT122 was raised against a GST-fusion protein of the WW domain of human FE65 (amino acids 253–285 of GENBANK/EBI Data Bank accession number O00213). This antibody specifically recognizes an 
97 kDa FE65 protein that appears in brain lysates, as same as reported previously (Kesavapany et al. 2002) (Fig. S3a). The specificity of UT122 was shown by immunostaining of mouse neuroblastoma Neuro 2a (N2a) cells expressing HA-tagged FE65 with UT122 and an anti-HA antibodies. The proteins recognized by the two antibodies were completely co-localized (Fig. S3b). Other antibodies used in this study were the mouse monoclonal antibodies anti- tubulin (TU-01, Invitrogen-Zymed), anti-microtubule associated protein 2 (MAP2) (HM-2, Sigma), anti-actin (MAB1501, Chemicon), anti-HA (12CA5, Roche), and anti-FLAG (M2, Sigma); the rabbit polyclonal antibodies anti-Thr668 APP phosphorylation state-specific (#2451, Cell Signaling Technologies), anti-Thr668 APP phosphorylation state-specific (raised against a synthetic peptide composed of AAV(pT)PEERC, IBL, Fig. S3c), anti-APP C-terminus (APP/C, Sigma), and anti-histone H1 (Santa Cruz Biotechnology); and the secondary antibodies goat polyclonal anti-rabbit and anti-mouse immunoglobulin conjugated to horseradish peroxidase (Amersham Pharmacia Biotech).
Subcellular fractionation of mice brains and cultured cells, and protein analysis
C57BL/6 mice brains or cultured cells were homogenized in eight volumes of buffer H (20 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA) (Gu et al. 2001) containing 1 µM microcystin-LR, 5 µg/mL chymostatin, and 5 µg/mL leupeptin and centrifuged at 2500 g for 10 min at 4 °C. The pellets were resuspended in buffer HB (10 mM HEPES-NaOH (pH 7.4), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 µM microcystin-LR, 5 µg/mL chymostatin, and 5 µg/mL leupeptin) and incubated on ice for 15 min. To 100 µL of this suspension, 6.25 µL of 10% (v/v) Nonidet P-40 was added, followed by agitation for 10 s and centrifugation at 10 000 g for 1 min. The pellet (crude nucleus fraction) was washed 3 times with buffer HB containing 0.625% (v/v) Nonidet P-40 and resuspended in a buffer consisting of 1% (wt/vol) SDS, 10 mM Tris-HCl (pH 7.5), 1 µM microcystin-LR, 5 µg/mL chymostatin and 5 µg/mL leupeptin. The sample was sonicated and the clear supernatant (nuclear fraction) was analyzed by immunoblotting. To obtain the membrane fraction, the supernatant of the buffer H homogenate was centrifuged at 100 000 x g for 1 h, after which the pellet was resuspended in buffer H containing 1% (wt/vol) SDS, 1 µM microcystin-LR, 5 µg/mL chymostatin, and 5 µg/mL leupeptin. The supernatant was used as the cytosolic fraction. The protein amounts in the fractionated samples were determined with the BCA protein assay kit (Pierce) and equal amounts of proteins were separated by electrophoresis in a Tris-glycine SDS-polyacrylamide discontinuous gel (8% (wt/vol) and 15% (wt/vol) polyacrylamide) or Tris-tricine gel (16% (wt/vol) polyacrylamide). The proteins were then transferred onto a nitrocellulose membrane, probed with the indicated antibodies, and detected using an ECL detection system (Amersham Pharmacia Biotech).
Immunoblot assays
Proteins except for AICD were detected as follows. N2a cells (1 x 106) were transiently transfected with the indicated amount of plasmid in Lipofectamine 2000 (Invitrogen) and cultured for 24 h in DMEM containing 10% (v/v) calf serum. The cells were harvested and lyzed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl containing 0.1% (wt/vol) SDS, 0.5% (wt/vol) sodium deoxycholate, 1% (v/v) Nonidet P-40, 150 mM NaCl) containing 5 µg/mL chymostatin, 5 µg/mL leupeptin, and 1 µM microcystin-LR for 0.5 h at 4 °C. Proteins in lysates were then separated by electrophoresis, transferred on to a nitrocellulose membrane, and probed with the indicated antibody. Reactive proteins were detected using an ECL detection system.
Detection of AICD in mouse brain and cultured cell
AICD in mouse brain was detected by immunoblotting as described (Gu et al. 2001) with some modifications. Equal amounts (300 µg) of the proteins in the fractions described above were subjected to immunoprecipitation with G369 in CHAPS lysis buffer (PBS containing 10 mM CHAPS, 5 µg/mL chymostatin, 5 µg/mL leupeptin, 1 mM Na3VO4, 1 mM NaF, and 1 µM microcystin-LR). The immunocomplexes were recovered with protein G-Sepharose at 4 °C. In a separate study, the immunoprecipitates were subjected to dephosphorylation with 400 units of lambda protein phosphatase (PPase) (Sigma) for 2 h in a Sigma-supplied buffer. These samples were then separated by electrophoresis in a Tris-tricine gel (16% (wt/vol) polyacrylamide). The separated proteins were transferred onto a nitrocellulose membrane, boiled in PBS for 5 min, and probed with an anti-APP cytoplasmic domain or Thr668-phosphorylation state-specific antibody. Reactive proteins were detected using an ECL-plus detection system. For AICD detection in cultured cells, we performed the same procedure described above without immunoprecipitation.
Plasmid construction
A cDNA encoding AICD, the sequence between amino acids 652 and 695 of human APP695, was produced by PCR using the primers 5'-cccgggatcccgccgggccagtacacatccat-3' (forward) and 5'-ggcggcggtaccgctagttctgcatctgct-3' (reverse) with the pcDNA3-FLAGAPP695 template (Ando et al. 1999). The AICD cDNA bearing a linker sequence (NH2-EFPGIPPG-COOH) at its 5'end (Cao & Sudhof 2001) was then cloned into the pBIND (Promega) vector at KpnI/BamHI sites, thus generating pBINDGal4BD-AICD, which encodes a fusion protein composed of a Gal4 DNA-binding domain (Gal4BD) linked to AICD. The region composed of the extracellular and transmembrane domains of APP695 (amino acids 1–651) was amplified with forward (5'-taatacgactcactatag-3') and reverse (5'-gggcccgctagctttcttcttcagcatcac-3') primers using pcDNA3APP695 (Tomita et al. 1999) as the template. The fragment was then cloned into the pGEM-T Easy vector (Promega), digested with HindIII and NotI and recloned into pcDNA3 with the same restriction sites to produce pcDNA3APPN. The inserted region of the pBINDGla4BD-AICD construct, from the start site of the Gal4-DNA-binding domain through to the synthetic polyadenylation signal, was amplified with forward (5'-gggcccgctagcatgaagctactgtcttct-3') and reverse (5'-cgatccttatcgctatcgat-3') primers, and the product was also cloned into pGEM-T Easy vector. The clone was then digested with NheI and NotI and recloned into pcDNA3APPN to generate pcDNA3APPNGal4BD-AICD plasmid. Plasmids encoding NFLAG-APPwt, NFLAG-APPT668A, NFLAG-APPT668E, FLAG-FE65, NHA-FE65, NHA-JNK11, and NHA-X11L have been previously described (Ando et al. 2001; Taru & Suzuki 2004).
Gene transactivation assay
Mouse neuroblastoma Neuro-2a (N2a) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). These cells were plated in 96 well plates (3 x 104 cells) and transiently transfected with various combinations of the following plasmids in Lipofectamine 2000 (Invitrogen): pBINDGal4BD-AICD (4 ng), pG5luc (40 ng) (Promega) and the indicated amount of pcDNA3.1NFLAG-FE65 or pcDNA3.1NFLAG. The cells were harvested 24 h after transfection and the transcriptional activity of the reporter gene was analyzed by using the dual luciferase assay system (Promega).
Immunofluoresence staining
N2a cells cultured on an 8-well chamber slide glass (Nalge Nunc International) were transfected with the indicated plasmids in Lipofectamine 2000 for 24 h, and the cells were fixed with 3.7% (w/v) formaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min, and blocked with PBS containing 3% (w/v) bovine serum albumin. The cells were rinsed with PBS and incubated with the indicated antibodies overnight at 4 °C, followed by incubation with goat anti-rabbit IgG coupled with Alexa Fluor 488 or goat anti-mouse IgG coupled with Alexa Fluor 546 secondary antibodies (Molecular Probe). After incubation with secondary antibody, cells were treated with diamidino-2-phenylindole (DAPI) solution for 5 min followed by washing for 3 times with PBS and observed under the confocal laser scanning microscope LSM510 (Carl Zeiss).
Mutant mouse
A 13-kb genomic fragment from C57BL/6 mice containing exons 17 and 18 was used to construct targeting vectors. The Thr668Ala point mutation was introduced by PCR mutagenesis. Mutant mice were isogenically generated using ES cells derived from same strain. The mutant mouse did not show any remarkable alterations of metabolism or distribution of APP (K. Seki, S. Takeda, Y. Sano, E. Kawaguchi, T. Nakaya, T. Suzuki and S. Itohara, unpublished observations).
Statistical analysis
For statistical analyses, we applied the non-parametric Tukey's test (Steel's method). Differences were judged to be significant if the P-value was < 0.05. Significance is indicated in figures with an asterisk (*).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tsuzuki{at}pharm.hokudai.ac.jp
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-secretases generate AICD. The AICD stays on the membrane and some population of AICD translocates into nucleus by FE65-independent yet unidentified mechanism. FE65 is anchoring to the cytoplasmic domain of APP. (Right) When APP was phosphorylated at Thr668 by some reason such as intracellular signal activation, FE65 is released from membrane, translocates into nucleus, and is trapped there by association with AICD.