The Intracellular Domain of the β-Amyloid Precursor Protein Is Stabilized by Fe65 and Translocates to the Nucleus in a Notch-like Manner

The β-amyloid precursor protein (APP) is a ubiquitous receptor-like molecule without a known function. However, the recent recognition that APP and Notch undergo highly similar proteolytic processing has suggested a potential signaling function for APP. After ligand binding, Notch is cleaved by the ADAM-17 metalloprotease followed by an intramembrane cleavage mediated by γ-secretase. The γ-secretase cut releases the Notch intracellular domain (NICD), which enters the nucleus and modulates transcription. Because APP is processed similarly by ADAM-17 and γ-secretase, we reasoned that the APP intracellular domain (AICD) has a role analogous to the NICD. We therefore generated a plasmid encoding the AICD sequence and studied the subcellular localization of the expressed protein (C60). Our results demonstrate that the cytoplasmic domain of APP is a highly labile fragment that is stabilized by forming complexes with Fe65 and can then enter the nucleus in neurons and non-neural cells. These findings strongly support the hypothesis that APP signals in the nucleus in a manner analogous to the function of Notch. The β-amyloid precursor protein (APP) is a ubiquitous receptor-like molecule without a known function. However, the recent recognition that APP and Notch undergo highly similar proteolytic processing has suggested a potential signaling function for APP. After ligand binding, Notch is cleaved by the ADAM-17 metalloprotease followed by an intramembrane cleavage mediated by γ-secretase. The γ-secretase cut releases the Notch intracellular domain (NICD), which enters the nucleus and modulates transcription. Because APP is processed similarly by ADAM-17 and γ-secretase, we reasoned that the APP intracellular domain (AICD) has a role analogous to the NICD. We therefore generated a plasmid encoding the AICD sequence and studied the subcellular localization of the expressed protein (C60). Our results demonstrate that the cytoplasmic domain of APP is a highly labile fragment that is stabilized by forming complexes with Fe65 and can then enter the nucleus in neurons and non-neural cells. These findings strongly support the hypothesis that APP signals in the nucleus in a manner analogous to the function of Notch. β-amyloid precursor protein Alzheimer's disease APP intracellular domain amyloid β-protein Notch intracellular domain CBP/Suppressor of Hairless/Lag-2 protein polyacrylamide gel electrophoresis Chinese hamster ovary 4,6-diamidino-2-phenylindole carboxyl-terminal fragment protein containing a disintegrin and a metalloprotease N-tris(hydroxymethyl)methylglycine The β-amyloid precursor protein (APP)1 is a type I transmembrane protein that is proteolytically processed by three enzymatic activities (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). The large ectodomain is first cleaved at one of three sites close to the membrane by either α- or β-secretase to liberate a soluble APP extracellular piece (termed α- or β-APPs). In doing so, α-secretase generates an 83-residue carboxyl-terminal fragment (CTF) C83, whereas β-secretase cuts at the other two sites to generate an 89- or 99-residue CTF (C89 and C99, respectively) (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar, 2Buxbaum J.D. Thinakaran G. Koliatsos V. O'Callahan J. Slunt H.H. Price D.L. Sisodia S.S. J. Neurosci. 1998; 18: 9629-9637Crossref PubMed Google Scholar). These 3 CTFs are retained in the membrane and become substrates for an unusual intramembrane cleavage mediated by γ-secretase, producing a heterogeneous set of products. The best characterized of these is the amyloid β-protein (Aβ) derived from C99, which accumulates to high abundance in senile plaques and appears to play a central role in the etiology of Alzheimer's disease (AD) (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). Whereas the several Aβ species have been studied in great detail, the other products generated by γ-secretase have received scant attention. One fragment of particular interest is theAPP intracellular domain (AICD), the ∼6-kDa extreme C terminus of APP that results from the γ-secretase cleavage of the C83, C89, or C99 fragments. The potential importance of AICD has recently been emphasized by the recognition of similarities between APP and another type I transmembrane protein, Notch (3Wolfe M.S. Haass C. J. Biol. Chem. 2001; 276: 5413-5416Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Notch is a cell surface receptor that plays a critical role in many cell fate decisions during embryogenesis and adulthood (4Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4951) Google Scholar). Following binding to its prototypical ligand Delta, Notch undergoes two cleavages, the first of which is performed by the metalloprotease ADAM-17, also called tumor necrosis factor-α converting enzyme (5Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israel A. Mol. Cell. 2000; 5: 207-216Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar, 6Mumm J.S. Schroeter E.H. Saxena M.T. Griesemer A. Tian X. Pan D.J. Ray W.J. Kopan R. Mol. Cell. 2000; 5: 197-206Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). It is subsequently cut by the presenilin-dependent γ-secretase (7De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Scchrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1808) Google Scholar), releasing theNotch intracellular domain (NICD), a fragment analogous to AICD. After release from the membrane, the NICD fragment associates with the cellular factor CSL (for CBF1,Suppressor of Hairless and Lag-1) and translocates into the nucleus, where it alters gene transcription (8Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar). APP is the only other known protein that shares similar sequential processing by ADAM-10 (9Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (986) Google Scholar) or ADAM-17 (10Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar) and γ-secretase (3Wolfe M.S. Haass C. J. Biol. Chem. 2001; 276: 5413-5416Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The striking similarities between Notch and APP proteolysis therefore raise the intriguing possibility that APP is a cell surface receptor that also signals via release and translocation of AICD into the nucleus. In the absence of a defined functional assay for APP in vitro or in vivo, we reasoned that an analysis of exogenous AICD expression could provide insights into its potential role in transducing a signal from the cell surface. However, detection of free AICD in intact cells has been elusive, similar to the situation with NICD. Because the binding of the CSL protein to NICD stabilizes the latter (8Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar), we hypothesized that an analogous mechanism might also apply for AICD. We therefore assessed whether a previously described protein, which binds to the APP cytoplasmic tail, Fe65, could stabilize AICD and allow nuclear translocation. Our results show that AICD complexes with and is stabilized by Fe65 in a manner analogous to the NICD and CSL proteins and that the complexes can enter the nucleus in both primary neurons and non-neural cells. A cDNA encoding an initiating methionine and the last 59 residues of the APP C terminus was amplified by polymerase chain reaction, inserted into pcDNA5 (Invitrogen), and confirmed by sequencing. Plasmids encoding wild-type human APP695 (11Selkoe D.J. Podlisny M.B. Joachim C.L. Vickers E.A. Lee G. Fritz L.C. Oltersdorf T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7341-7345Crossref PubMed Scopus (543) Google Scholar) and human Fe65 with a Myc epitope (12Lau K.F. McLoughlin D.M. Standen C.L. Irving N.G. Miller C.C. Neuroreport. 2000; 11: 3607-3610Crossref PubMed Scopus (43) Google Scholar) have been characterized. For transfections, 10 µg of DNA was introduced into COS cells (95% confluent) or mouse primary neurons (18,000 cells/cm2) using LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's instructions. Neurons were plated and maintained in neurobasal media supplemented with B27 (Life Technologies, Inc.) using standard methods (13Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar). Polyclonal antibodies C7 and X81 were described previously (Refs. 14Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar and 15Podlisny M.B. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E.H.M. Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google Scholar, respectively). Monoclonal antibodies 13G8 and 8E5 were gifts of P. Seubert and D. Schenk and are directed against APP732–751 and APP500–648, respectively. Polyclonal antibody C4 is raised against APP722–751 and was a gift of Y. Ihara. Monoclonal antibodies 9E10 (which recognizes the c-Myc epitope) and histone H1 were purchased from Santa Cruz Biotechnology, and polyclonal tubulin antibody was from Sigma. De novo generation of Aβ and AICD was performed essentially as described (16Xia W. Ray W.J. Ostaszewski B.L. Rahmati T. Kimberly W.T. Wolfe M.S. Zhang J. Goate A.M. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9299-9304Crossref PubMed Scopus (132) Google Scholar). The membrane pellet (microsomes) was resuspended in 50 mm Tris, pH 7.0, 150 mm NaCl, 1 mm EDTA. The resuspended vesicles were incubated for 4 h at 37 °C, and the reaction stopped with Nonidet P-40 at a final concentration of 1%. Experiments were performed in 3 independent replicates. The nuclear fractionation of whole cells was performed as described (17Thomas J.E. Smith M. Rubinfeld B. Gutowski M. Beckmann R.P. Polakis P. J. Biol. Chem. 1996; 271: 28630-28635Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Following Dounce homogenization, nuclei were pelleted at 800 × g and washed three times in wash buffer containing 0.1% Nonidet P-40. In the postnuclear supernatant, membranes were separated from cytosol with a 100,000 × g spin for 45 min. Cells were solubilized in 100 µl of an SDS lysis buffer containing 50 mm Tris, pH 7.6, 150 mm NaCl, 2 mmEDTA, 1% SDS, and Complete protease inhibitors (Roche Molecular Biochemicals). Lysates were sonicated at 4 °C for three 5-s pulses and then diluted 1:9 with a detergent buffer identical to 1% SDS lysis buffer except that it contained 1% Nonidet P-40 instead of SDS. Some samples were lysed directly in 1% Nonidet P-40 lysis buffer. Lysates were precipitated with C7 (for APP, 1:200) or 9E10 (for Fe65, 1:100) and 50 µl of protein A-Sepharose (Sigma) or protein G-agarose (Roche) for more than 2 h at 4 °C. Following extensive washing, samples were eluted in 2× sample buffer (20% glycerol, 4% SDS, 10% β-mercaptoethanol) at 100 °C for 5 min, resolved by 10–20% Tris/Tricine SDS-PAGE (Bio-Rad), and transferred to 0.2 µm polyvinylidine difluoride (Bio-Rad). Western blotting using ECL Plus detection was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). All experiments were repeated at least three times. Cells plated on 18-mm coverglass were fixed for 15 min in 4% paraformaldehyde and then blocked with 5% goat serum in phosphate-buffered saline containing 0.1% Triton X-100 for 1 h. Primary antibodies (C7, 1:2000; C4, 1:2000; 9E10, 1:1000) were diluted in the blocking buffer and incubated for 2 h. Following three 5-min washes with phosphate-buffered saline, secondary antibodies (goat anti-rabbit Cy3, 1:400; goat anti-mouse FITC, 1:200; Jackson ImmunoResearch) were incubated for 1 h at room temperature. Nuclei were stained with 1 µm DAPI for 30 min and mounted with SlowFade Light anti-fade reagent (Molecular Probes). Images were collected with an Axiovert 100M confocal scanning microscope and analyzed with LSM 510 software (Zeiss). The APP intracellular domain (AICD) corresponds to the cytoplasmic tail of APP that is released after cleavage of C83, C89, or C99 by γ-secretase (see Fig. 1a). In our search for the AICD fragment, we first attempted to generate it using a cell-free in vitro reaction. We had previously measured Aβ generation in microsome vesicles derived from Chinese hamster ovary (CHO) cells stably overexpressing APP751 (16Xia W. Ray W.J. Ostaszewski B.L. Rahmati T. Kimberly W.T. Wolfe M.S. Zhang J. Goate A.M. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9299-9304Crossref PubMed Scopus (132) Google Scholar). We therefore probed for de novo generation of AICD by similarly incubating microsomes at 37 °C. After the incubation, the microsomal membranes were lysed, precipitated with the APP antiserum C7, and resolved by 10–20% Tris/Tricine SDS-PAGE. Identical preparations incubated in parallel were analyzed by a sensitive and specific sandwich enzyme-linked immunosorbent assay for Aβ, novo of Aβ in 4 In these we the de novo of a fragment of APP that was only after the at 37 °C of fragment was by a characterized of γ-secretase, M.S. Xia W. C.L. Ostaszewski B. Rahmati T. Selkoe D.J. 1999; PubMed Scopus Google Scholar) 1 3 and that the Tris/Tricine gel the three APP CTFs C89, and which are more in a 1 We a plasmid encoding the 59 of APP an initiating methionine (C60). The expressed protein corresponds to the APP sequence after the γ-secretase cleavage after of We expressed in COS cells and lysed in 1% Nonidet P-40 followed by 1% SDS. In COS an fragment to was in the Nonidet and only a was in the Nonidet 1 2 and In the COS a ∼6-kDa was in the Nonidet the of the protein was in the Nonidet pellet 1 3 and Because SDS, Nonidet we that of the protein was with the nuclei of these cells. the subcellular localization of the expressed protein, we performed a nuclear fractionation described previously (17Thomas J.E. Smith M. Rubinfeld B. Gutowski M. Beckmann R.P. Polakis P. J. Biol. Chem. 1996; 271: 28630-28635Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Following we the COS cells to Dounce and pelleted After the nuclear with 0.1% Nonidet P-40 to the nuclei were lysed in 1% SDS. The postnuclear were at 100,000 × g to membranes from The localization of three protein confirmed the of the fractionation 1 We therefore to the of in COS cells by and Western APP to and C83, to the membrane as 1 and confirmed that or membrane protein the nuclear In cells with a of the expressed protein was to the nucleus 1 with a with the membrane Because the CSL protein is known to stabilize the NICD fragment (8Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar), we reasoned that one of the APP cytoplasmic binding might a similar function for APP. are several proteins that are to to APP Strooper B. Annaert W. J. Sci. 2000; PubMed Google Scholar), we our on Fe65 it is a prototypical of a of that alters APP processing O. S. P. J.D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. Haass C. A. J. 1999; PubMed Scopus Google Scholar, G. de P. A. R. N. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), to the nucleus G. de P. A. R. N. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and binds to the transcription factor N. G. de P. T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). we with Fe65, we a in in three subcellular 1 Fe65 was in the three subcellular in with previously G. de P. A. R. N. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). However, AICD was Fe65 was 1 In the of Fe65 with APP695 allow of an AICD fragment that the latter is with great and by more directly the subcellular localization of we confocal to intact cells. was we were to cells that C7 the and in parallel and analyzed by blotting confirmed expression of that confocal is sensitive than Western Because expression of the protein than of and Fe65 (see we with We cells for the C terminus of APP and Fe65 followed by with DAPI to these specific for was to the nucleus was also in the Fe65 was 2 and signal was with the C7 the two to a great 2 was with a APP C4 for the of we C7 with its which the without Fe65 detection the C7 and Fe65 primary antibodies were we and we confirmed that Fe65 subcellular localization is independent of it retained the expression in the or absence of We assessed whether Fe65 can to the that Fe65 binds to the C terminus of APP N. J.D. G. F. De P. De S. R. S. J. M. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore performed in COS cells both and Fe65 via its Myc and then Western blotting for APP with C7 a only the two proteins were 2 and was confirmed the of the antibodies was with an antibody and Western blotting with an antibody Fe65 the two proteins were we also of Fe65 it was expressed Because the antibody also recognizes APP, we to that a of Fe65 with APP. In we that C7 precipitated APP in three these that Fe65 binds to APP and demonstrate that is maintained the fragment is the of Fe65 on we performed COS cells were incubated with for 10 min and in media for followed by of the in we followed by Fig. 3 the results from three independent an in the of expressed A of was with Fe65 We its to min expressed min Fe65 was also the latter that a of the protein is more at times in the of APP is expressed in many in the it is in neurons (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar, A. A. F. D. De C. R. F. F. T. Neurosci. PubMed Scopus Google Scholar). Fe65 is also expressed in the A. N. R. F. T. 19: PubMed Scopus Google Scholar), that the and of these two proteins in We therefore whether protein was into primary We the or mouse neurons with or without and analyzed the by and Western blotting 3 The results were similar to with COS cells. the of in expression 3 with Fe65 in the of a The in the of in COS cells and neurons is to the in these two cell as in COS Fe65 stabilizes the Because the C terminus of APP is known to in neurons J.D. Thinakaran G. Koliatsos V. O'Callahan J. Slunt H.H. Price D.L. Sisodia S.S. J. Neurosci. 1998; 18: 9629-9637Crossref PubMed Google Scholar), we also several CTF to the α- and β-secretase and C89, in both and the subcellular localization of in we performed nuclear fractionation on the with the COS we APP and to in the membrane the of our fractionation 3 of and Fe65 and fractionation to the detection of the ∼6-kDa fragment in three subcellular 3 in with the COS results 1 we nuclear in the the importance of Fe65, we whether expression of protein the detection of AICD derived from APP. we a cell that stably APP751 with the M.B. Ostaszewski B.L. Koo E.H. Teplow D.B. Selkoe D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with or was a ∼6-kDa was 3 which was stabilized by Fe65 we similar results Fe65 was into primary mouse which of APP695 3 4 In we provide that the hypothesis that the free APP intracellular domain (AICD) by γ-secretase cleavage is stabilized by and the nucleus with the protein, The potential importance of the that our experiments in intact cells is by its striking to CSL and NICD in Notch APP and Notch are cleaved of the membrane by an α-secretase mediated by These release the the to cleaved by γ-secretase. cleavage is highly unusual and is by other proteins M.S. Ye J. J.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The highly labile NICD fragment then binds to the CSL protein and translocates into the nucleus where it the transcription of The detection of NICD is by the of CSL (8Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar). Our results that and Fe65 have an analogous is highly labile and binds Fe65, is in the and nucleus. Fe65 stabilizes and nuclear translocation of in a manner similar to CSL and NICD. our results support an role for Fe65, other binding also AICD other binding can APP B. R.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It is that a several proteins of binding the cytoplasmic domain of APP on its proteolytic fate and function. Our findings in are with recent in cell that the cytoplasmic domain of APP can in which transcription of in the nucleus X. T. Science. 2001; PubMed Scopus Google Scholar). Our that the free cytoplasmic domain generated by γ-secretase cleavage from APP is in cells after by Fe65 the by and X. T. Science. 2001; PubMed Scopus Google Scholar) that sequential α- and γ-secretase to release the cytoplasmic domain to signal in the nucleus. the a mechanism of APP function that could previously for APP in and E.A. R. D. K. C.L. 1992; Full Text PDF PubMed Scopus Google Scholar, A. Miller C. Koo E.H. Selkoe D.J. J. Neurosci. PubMed Google Scholar, Koo E.H. J. Neurosci. 1997; PubMed Google Scholar). In our of the APP cytoplasmic domain are with that a primary function of APP as a receptor that signals cleavage and nuclear translocation of its cytoplasmic tail with proteins that a mechanism striking to that of the Notch These two with a protein, to a of receptor that signal cleavage transmembrane We Y. for the gift of antibody C4 and D. McLoughlin for the Fe65 We are to S. Vasquez for with the mouse and are to D. M. and M. Schlossmacher for