The β-amyloid precursor protein APP functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation

Giuseppina Minopoli, Paola de Candia, Alessandro Bonetti, Raffaella Faraonio, Nicola Zambrano, and Tommaso Russo§

Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy

§ To whom correspondence should be addressed:Tommaso RussoDipartimento di Biochimica e Biotecnologie MedicheUniversità di Napoli Federico IIVia S. Pansini 5, I-80131 Naples, ItalyPhone: +390817463131FAX: +390817464359E-mail: russot@dbbm.unina.it

Running title: Fe65 nuclear localization

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARYIn this study we addressed the question of the intracellular localization of Fe65, an adaptor protein interacting with the β-amyloid precursor protein (APP) and with the transcription factor CP2/LSF/LBP1. By using tagged-Fe65 expression vectors, we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolation of nuclei from untransfected PC12 cells allowed us to observe that a part of the endogenous Fe65 is present in the nuclear extract. The analysis of Fe65 mutant constructs demonstrated that the region of the protein required for its nuclear translocation includes the WW domain, and, on the other hand, a small fragment of 100 residues, including this WW domain, contains enough structural information to target a reporter protein (GFP-GFP) to the nucleus. To evaluate whether the Fe65-APP interaction could affect the Fe65 intracellular trafficking, COS7 cells were co-transfected with APP695 or APP751 and with GFP-Fe65 expression vectors. These experiments demonstrated that Fe65

is no longer translocated to the nucleus when the cells overexpress APP, while the nuclear targeting of GFP-Fe65 mutants, unable to interact with APP, is unaffected by the co-expression of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol.

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INTRODUCTIONThe β-amyloid precursor protein (APP) is a widely expressed integral membrane protein that is involved in the pathogenesis of Alzheimers diseases (AD). In fact, the main constituent of the AD senile plaques, the β-amyloid peptide (Aβ), derives from APP as a consequence of its proteolytic processing (for a recent review see ref. 1). The overall structure of APP resembles that of a receptor or growth factor precursor, but only a small fraction of APP resides at the plasma membrane (2, 3). Part of the protein reaching the cell surface, through the secretory pathway, undergoes a juxtamembrane cleavage by the action of the α-secretase-ADAM10 protease (4) that results in the generation of a secreted, soluble form of APP. The rapid turnover of surface APP drives the protein to the endocytic compartment and then again to ER-Golgi network (5, 6), where it undergoes the cleavage by another proteolytic activity named β-secretase-BACE-1 (7-10), which generates a transmembrane 12 kDa fragment. This fragment is then cleaved by the γ-secretase activity which leads to the formation of Aβ. Recent evidence strongly suggests the identity of γ-secretase and presenilin dimer (11, 12). The elucidation of the normal functions of APP could be important to understand the molecular basis of AD and to approach its prevention and/or treatment, but it has been up to now elusive. One of the characteristics of this protein that is currently investigated is the complex network of protein-protein interactions that is centred at the short cytoplasmic tail of APP (13, 14). Many proteins, in fact, interact with this C-terminal domain of APP, most of them possessing multiple protein-protein interaction domains which in turn form complexes with other proteins, that suggests they function as adaptor proteins bridging APP to specific molecular pathways. Three of these adaptor proteins belongs to the Fe65 protein family; Fe65 has been originally described as a protein highly expressed in neurons of specific areas of the mammalian nervous system and also possessing some characteristics of a transcription factor (15, 16). It contains three protein-protein interaction domains, a WW and two PTB domains. The PTB2 domain, located in the C-terminal half of the molecule, is responsible for the interaction of Fe65, and of the two related proteins Fe65L1 (17) and Fe65L2 (18), with the cytosolic tail of APP (19-21). Similarly, all the three members of the Fe65 family interact with two APP related proteins, APLP1 and APLP2 (17, 18). These interactions take place at the level of the ΦXNPXY motif (where Φ indicates an hydrophobic residue and X any amino acid) present in the cytodomains of APP and of its related proteins, and, differently from that observed for other PTB domains, the interaction does not require the phosphorylation of the tyrosine residue (20, 22). Other molecular adaptors, possessing a single PTB domain, bind to the same ΦXNPXY motif of APP: the proteins belonging to the X11 protein family (20, 23-25) and m-Dab1 (26, 27), the mammalian orthologue of the product of the disabled gene of Drosophila. The formation of these complexes seems to affect the regulation of APP processing, considering that the expression of Fe65 leads to an increased generation of Aβ in cultured cells (28, 29), while X11-APP co-expression inhibits the proteolytic processing of APP (25, 30, 31). Lastly, APP seems to have also other partners, not possessing a PTB domain, that bind its cytodomain, such as a Go protein (32), and two more proteins, named PAT-1 (33) and APP-BP1 (34).This scenario appears even more complex, when the molecular partners of the APP ligands are examined. The WW domain of Fe65, in fact, interacts with several proteins, one of which is Mena, the mammalian orthologue of the product of the enabled gene of Drosophila, which is a genetic suppressor of the phenotype induced in Drosophila by the abl gene mutation (35). The second PTB domain of Fe65 (PTB1) interacts with the transcription factor CP2/LSF/LBP1 (36) and, at least in vitro, with the LDL-receptor related protein LRP (27). On the other hand, particularly at the presynaptic level, X11 forms a trimeric complex in vivo with CASK and VELI, the orthologues of C. elegans Lin2 and Lin7 protein, respectively (37-39), and interacts with Munc18 (40) and with a

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calcium ion channel (41). Considering that the intracellular trafficking of APP appears to be relevant for the different proteolytic fate of the molecule and, possibly, for its function(s), it is important to analyze the intracellular localization of proteins interacting with the cytodomain of APP, to evaluate the possible association of any APP partner to specific APP trafficking and proteolytic pathways. The ligands of Fe65 up to now identified suggest multiple intracellular locations of this adaptor protein, considering that Mena is mainly connected with the actin cytoskeleton remodelling system (42) and the known functions of CP2/LSF/LBP1 take place in the nucleus. In this paper we addressed the question of the intracellular localization of Fe65 and its relationship with that of APP. We found that transient overexpression of Fe65 in cultured cells results in its accumulation in the nucleus and that endogenous Fe65 is present in the nuclear extract from PC12 cells. The cytosol-nuclear translocation of Fe65 takes place through an unusual pathway requiring the region that includes the WW domain and is prevented by the co-expression of APP, whose cytodomain functions as a cytosolic anchoring element.

EXPERIMENTAL PROCEDURESGeneration of GFP-Fe65 expression vectorsThe GFP-Fe65 and Fe65-HA fusion proteins encoding wild-type Fe65 were obtained by amplification of the rat Fe65 cDNA previously described (43). The Fe65 deletion mutants GFP-Fe65∆666-711, GFP-Fe65∆538-711, GFP-Fe65∆1-287, GFP-Fe65∆1-253 as well as the fragment corresponding to the Fe65 cDNA codons 191-290 were obtained by amplification of the Fe65 cDNA with specific oligonucleotide primers (CEINGE) as follows: for GFP-Fe65 and Fe65-GFP, forward1: 5-GAGCTCAAGCTTCTACTAAGCCATGTCTGTTC and reverse1: 5-GACCGCGGGCCCGCGGGGTCTGGGATCCTAG; for GFP-Fe65∆666-711, forward1 and reverse2: 5-GCCATCGGGCCCAAGCATCCAGACACTTCTGGTA; for GFP-Fe65∆538-711, forward1 and reverse3: 5-GCCATCGGGCCCAAGCATTGCGCCTTCAGACA; for GFP-Fe65∆1-287, forward2: 5-GAGCTCAAGCTTCCCCATCACAGGGGAACAG and reverse1; for GFP-Fe65∆1-253, forward3: 5-GAGCTCAAGCTTCCGATCTACCGGCTGGATG and reverse1; for GFP-GFP-Fe65191-290, forward3: 5-ATATGAATTCTGGACCCCAGGYCCTCACAGATGG and reverse4: 5’-CTGCGAATTCTGATGGGGAGGCCCGGTCC. The recombinant constructs were generated by ligation of the polymerase reaction fragments digested with appropriate restriction enzymes and purified from agarose gels with the QIAEX gel extraction kit (QIAGEN) in the pEGFP-C1 and pEGFP-N1 vectors (Clontech) for expression as a fusion to the C-terminus and to the N-terminus of EGFP, respectively. The GFP-GFP expression vector was generated by cloning in the XhoI–EcoRI sites of the pEGFP-C1 vector (Clontech) the EGFP cDNA obtained by direct amplification from the same vector with the following oligonucleotide primers: forward4: 5’-ATATCTCGAGAGCTGGACGGCGACGTAAACG and reverse5: 5’-ATAAGAATTCAGGTAGTGGTTGTCGGGCAGC.The GFP-GFP-Fe65/191-290 was obtained by cloning in frame the corresponding cDNA fragment downstream the region encoding the second GFP in the GFP-GFP vector.

The Fe65 point mutant Cys655->Phe cDNA was obtained by using the Quick-changeTM kit (Stratagene), according to the manufacturer’s procedure. For the mutagenesis the pEGFP-C1-Fe65 expression vector was used as template and the following pair of oligonucleotides as primers:forward5: 5’-CTGTGCAGGCTGACATTCATGCTCCGCTAC and reverse6: 5’-GTAGCGGAGCATGAATGCAGCCTGCACAG. Bold characters indicate the mutated residue.The sequence and the reading frame of all the recombinant constructs were checked by nucleotide sequence by using the Sequenase kit (Amersham-Pharmacia Biotech.) and the expression and the size of the fusion proteins was confirmed by western-blot analysis.The CMV-Fe65-HA vector expressing wild type rat Fe65 tagged with the HA epitope was

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obtained by cloning in the HindIII site of pRC-CMV (Invitrogen) a PCR fragment obtained by using the following primers: forward 6: 5’-CCCAAGCTTACTAAGGCCATGTCTGTTCCA and reverse HA: 5’-CCCAAGCTTTCAAGCGTAATCTGGAACATCATATGGGTATGGGGTCTGGGATCCTAGAC.

Cell culture conditions, transfections and fluorescence microscopyCells were grown at 37° C in the presence of 5% CO2; COS-7 cells were cultured in DMEM (Life

Technologies Inc.) supplemented with 10% foetal bovine serum (HyClone) and 1% penicillin/streptomycin mixture (HyClone). PC12 cells were cultured in RPMI (Life Technologies Inc.), supplemented with 10% Horse serum (Gibco BRL), 5% foetal bovine serum (HyClone) and and 1% penicillin/streptomycin mixture (HyClone). For the expression of the GFP-Fe65 fusion

proteins or of Fe65-HA, 1,5 x 106 cells were transfected by electroporation at 250 microfarad and 220 V with 20 µg of each construct. For co-transfection experiments 10 µg each of the GFP-Fe65 constructs and of the CMV-APP expression vectors, carrying the wild-type APP695 or the

APP751 cDNAs, were used.

For fluorescence microscopy, transfected COS-7 cells were grown on coverslips. After 36 hrs cells were washed with PBS and fixed with paraformaldehyde (4% in PBS pH 7.4) for 20 min at room temperature. The cells were then washed once in PBS-glycine and twice in PBS. When indicated, the cells were permeabilized with 0.1% Triton X-100. APP was stained with 6E10 or 369 antibodies, using Texas red-conjugated secondary antibodies (Jackson ImmunoResearch Lab.). Fe65-HA was stained with the anti-HA polyclonal antibody Y-11 (Santa Cruz Biotech.). The coverslips were mounted in 50% glycerol solution onto a glass microscope slide and viewed using an Axiophot microscope (Zeiss). For fluorescence observation of unfixed cells the coverslips were lied down on a drop of PBS and immediately examined. Confocal microscopy analysis was performed on a laser scanning LSM510 microscope (Zeiss) using dedicated image software.

Extract preparation, immunoprecipitation and western blot analysisFor the preparation of total cell extracts, monolayer cultures were harvested in cold PBS and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml each of aprotinin leupeptin and pepstatin). The extracts were clarified by centrifugation at 16,000 x g at 4° C and the protein concentration was determined by the Bio-Rad assay according to manufacturers instructions. 10 µg of extract were analyzed by SDS-PAGE followed by immunoblotting as described (22). For the immunoprecipitation experiment, the cell extracts (2mg) were incubated with the monoclonal antibody anti-GFP JL8 (Clontech) according to the manufacturers instructions. The immunocomplexes were collected with 30 µl/sample of protein A/G plus-Agarose (Santa Cruz Biotechnology), resolved on SDS-PAGE gel and transferred to Immobilon-P membranes (Millipore).PC12 cell fractionation was performed as previously described (36) and nuclei were purified on a 30% sucrose cushion by centrifugation at 9,000 x g for 15 min at 4°C. Nuclear and cytosol-membrane extracts were prepared as described (36).

RESULTS AND DISCUSSIONIntracellular localization of Fe65In order to characterize the intracellular localization of Fe65 and to follow its possible intracellular trafficking, we generated two constructs driving the expression of full length Fe65 fused to the

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green fluorescent protein GFP, one having GFP at the N-terminus (GFP-Fe65) and the other one at the C-terminus (Fe65-GFP) of Fe65. These constructs were transfected in COS7 cells and the western blot analysis of transfected cell extracts demonstrated that both fusion proteins are stable and of the expected size (see Figure 1, panel A). The characteristic multiple band pattern of Fe65 (36) was also observed with GFP-Fe65 and Fe65-GFP; the latter migrates slightly faster than GFP-Fe65. The subcellular distribution of GFP-Fe65 fusion proteins in transfected cells showed that most of the fluorescent signal is localized in the area of the nucleus, with the exclusion of nucleoli (see Figure 1, panels B and C). Furthermore, Fe65-bound fluorescence is also present outside the nuclei and in many cells a clear definition of some edges can be seen. No difference was observed between the cells transfected with GFP-Fe65 or with Fe65-GFP. Figure 1 also shows that there is no significant difference between the localization of the fluorescent signal in fixed cells (panels B and C) and in living unfixed cells (panels D and E). The confocal microscope analysis confirmed that the fluorescence bound to Fe65 is present within the nuclei (see Figure 2).An identical expression pattern was seen when COS7 cells were transfected with the CMV-Fe65-HA vector, which drives the expression of wild type rat Fe65 protein tagged at the C-terminus with the hemagglutinin HA epitope, and stained with an anti-HA antibody (see Figure 1, panel F). Also in this case, the Fe65-HA signal is significantly represented both in the nucleus and in the cytosol.To evaluate whether also endogenous Fe65 is present in the nucleus of non transfected cells, we examined PC12 cells, in which the levels of the protein is significantly higher those present in COS7 cells. Nuclei were purified from exponentially growing PC12 cells as previously described (36) and the protein extract from these nuclei was examined by western blot. As shown in Figure 1, panel G, Fe65 is clearly present in the nuclear extract (N), while its amount in the cytosol-membrane (CM) fraction is several fold higher than in the N fraction. The WW-containing region is responsible for Fe65 nuclear translocationThe molecular weights, deduced from the DNA sequence, of the two GFP-Fe65 fusion proteins and that of Fe65-HA render their free diffusion through the nuclear pore unlikely, thus we addressed the question of what region(s) of Fe65 is(are) responsible for its nuclear localization. To this aim, we generated a series of GFP-Fe65 vectors expressing various deletion mutants of Fe65. These vectors, summarized in Figure 3, panel A, were transfected in COS7 cells and this resulted in the expression of fusion proteins of the expected sizes (see Figure 3, panel H). The amino acid sequence of rat Fe65 contains only one region, from amino acid 684 to 705, with clustered basic residues (RRX5RRX9KXKR) that resembles a nuclear localization signal (NLS) and is conserved

in the human protein. On this basis a construct was generated driving the expression of a GFP-Fe65 fusion protein containing a deletion mutant of Fe65, lacking the C-terminal 23 residues, from amino acid 666 to 711 (see Figure 3, panel A). Figure 3B shows that the cellular localization of this GFP-Fe65∆666-711 is identical to that of the GFP-Fe65wt protein, being localized mainly in the nucleus and also in the cytoplasm. On the contrary, the truncation of the N-terminal 287 amino acids of Fe65 results in a dramatic change of the intracellular distribution of the GFP-Fe65∆1-287 protein, that is completely excluded from the nucleus (see Figure 3, panel C). The same exclusion from the nucleus was also observed in living unfixed cells (see Figure 3, panel D). To further restrict the sequence involved in the Fe65 nuclear localization, we generated some more deletion mutants. As shown in Figure 3E, one of these mutants, GFP-Fe65∆1-253 (see Figure 3, panel A), is still translocated to the nucleus. Therefore, the analysis of the GFP-Fe65 deletion mutants restricts the area in which the element responsible for the Fe65 nuclear localization from amino acid 250 to amino acid 290, which includes the WW domain. These results are in agreement with the results of previous experiments based on cell fractionation, which demonstrated that Fe65 is present in both nuclear and cytosolic fractions, while deletion mutants of Fe65, lacking the N-terminal region, are only found in the cytosolic extracts (36).

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No typical NLS is present in this region, therefore, we addressed the question of the possible presence in this region of an element that, besides to be necessary for Fe65 nuclear localization, is also sufficient to target a protein to the nucleus. To do this we generated constructs encoding fusion proteins composed by two consecutive GFP units fused to various fragments of the region of Fe65 from amino acid 1 to 290 that we demonstrated to be necessary for its nuclear translocation (see Figure 3, panels A and H). As shown in Figure 3F a protein consisting of two consecutive GFP units is excluded from the nucleus, as expected from its molecular size, while the GFP-GFP-Fe65 protein carrying the region from amino acid 191 to 290 is translocated into the nucleus (see Figure 3, panel G). There are several possibilities to be examined to explain the nuclear translocation in the absence of a consensus NLS. One of these is the direct interaction of Fe65 with the nuclear pore complex, as observed for other nuclear proteins lacking a NLS (44). Another possibility is that Fe65 is transported to the nucleus by a cargo system including a protein containing NLS. An example of this “piggyback” mechanism is that of cyclin B1 that, lacking NLS, is translocated into the nucleus in a complex containing cyclin F, which possesses two functional NLS (45). We previously demonstrated that the WW domain of Fe65 binds several proteins, only one of which has been identified as Mena (35), which is not a good candidate to be responsible for the translocation of Fe65 to the nucleus, being mainly localized in the cytosol. The attempt to use the WW domain of Fe65 as a bait to find its partners by the two-hybrid system screening failed because of the high background given by the GAL4-Fe65 fusion protein. This background is expected, considering the previous observation that the region of Fe65 containing the WW domain is per se able to strongly activate the transcription when fused to the GAL4 DNA binding domain (16). This means that the identification of the other ligands of the WW domain of Fe65, possibly responsible for its cytosol-nuclear trafficking, will require their purification and sequencing by classical biochemical procedures. Figure 3G shows that, unlike the wild type GFP-Fe65 protein that is present both in the nucleus and in the cytosol (Figure 1), the GFP-GFP-Fe65191-290 is restricted to the nucleus. This suggests that a nuclear export signal (NES) present in Fe65, has been removed from the GFP-GFP-Fe65191-290 protein. One possible NES (LX3LX2VXV) present in Fe65 from residue 180 to 189

and absent from the GFP-GFP-Fe65191-290 protein, cannot be considered because it is also absent from the GFP-Fe65∆1-253 that is located both in the nucleus and in the cytosol (see Figure 3E). Thus, the possible sequence affecting Fe65 export from the nucleus should be located in the C-terminal half of the molecule or, as in the case of Fe65 nuclear import, it is translocated to the cytosol through the interaction with an exported protein. CP2/LSF/LBP1 interacts with the PTB1 domain of Fe65, thus could be involved in Fe65 nuclear export. However, the nuclear-cytosol transport of this transcription factor was never addressed and its sequence does not contain any canonical NES.

The overexpression of APP prevents the translocation of Fe65 to the nucleus and results in the accumulation of Fe65 at the plasma membrane and in the ER-Golgi areaThe protein-protein interaction network centred at the cytosolic domain of APP could be involved in transmembrane signalling triggered by the interaction of APP with soluble or cell-anchored signals. The demonstration that Fe65 does transit in the nucleus suggests that the Fe65-APP interaction could affect some nuclear functions. In this optics, we analyzed the possible relationship between Fe65 intracellular trafficking and APP expression. To address this point we co-transfected COS7 cells with GFP-Fe65 and APP695 expression vectors. Transfected cells were stained with

6E10 antibody which recognizes the extracellular/intraluminal domain of APP or with 369 antibody which recognizes the C-terminal cytosolic domain of APP. As shown in Figure 4, in non-permeabilized cells, 6E10 antibody allowed to stain the cells transfected with APP695 expression

vector (panel A) and some of these cells were at the same time expressing GFP-Fe65. In these co-

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transfected cells Fe65-bound fluorescence is excluded from the nucleus, while in the cells expressing only GFP-Fe65 the fluorescence is localized in the nucleus (see Figure 4, panel A’). The same experiment was done in permeabilized cells, by using the 369 antibody, which recognizes the cytosolic domain of APP. In these conditions, further than the already observed exclusion from the nucleus of GFP-Fe65wt in the cells co-expressing APP, a clear co-localization of GFP-Fe65wt and APP was seen at the plasma membrane, in the perinuclear cisternae and in the area of the Golgi network (see Figure 4, panels B and B’). The co-transfection of the GFP-Fe65 vector with APP751

expressing vector gave results identical to those observed with APP695 (data not shown).

These experiments demonstrated that Fe65 is no longer translocated to the nucleus when the cells overexpress APP, thus suggesting that the interaction with APP is sufficient to tether Fe65 in the cytosol. To support this hypothesis, we generated an expression vector which encodes a GFP protein fused to the deletion mutant of Fe65 (GFP-Fe65∆538-711, see Figure 3, panel A) that lacks the PTB2 domain which is responsible for the binding to APP. Figure 5 (panels A-A) shows that the GFP-Fe65∆538-711 mutant is located in the nucleus and in the cells co-expressing APP695

the nuclear translocation of the Fe65 mutant is unaffected. Furthermore, this GFP-Fe65∆538-711 mutant does not accumulate in the perinuclear cisternae and in the Golgi area. An identical behaviour was observed when the cells were transfected with a GFP-Fe65 construct bearing a point mutation changing the Cys655 of Fe65 into a Phe (see Fig. 5, panels B-B’). This Fe65 mutant is unable to interact with APP (20), as demonstrated by the coimmunoprecipitation experiment reported in Figure 5, panel C. In fact, APP was not co-immunoprecipitated with Fe65 in extracts from COS7 cells transfected with Fe65C655F and APP695, while APP co-immunoprecipitates with

wild type Fe65. The intracellular localization we observed in COS7 cells co-transfected with Fe65 and APP is very similar to that previously reported in MDCK-695 cells stably expressing Fe65 (28). In fact, Fe65 and APP are clearly co-localized at the level of perinuclear cisternae and in the Golgi area. In these cells transfected Fe65 was not seen in the nucleus. The reason for this difference probably is, as also suggested by the Authors of that study, that the amount of holo-APP in MDCK-695 cells is not limiting, therefore, the transfected Fe65 remains entrapped in the cytosol where a large amount of APP is present. In any case, it is possible that also in MDCK-695 cells a fraction of Fe65 is translocated to the nucleus, but the amount of this fraction could be below the detection limit of the immunomicroscopy procedure used. According to this possibility, in non-transfected PC12 cells the amount of endogenous Fe65 present in the nuclear extract is much more lower than that present in the cytosol-membrane fraction (see Figure 1, panel G). The inhibition of Fe65 nuclear import by the interaction with APP could be a new example of regulated nuclear localization by cytoplasmic anchoring. The best studied example of this phenomenon is that of β-catenin. This protein plays an important role in the Wnt-signalling pathway (46); it functions as an adaptor protein binding to cadherins, membrane proteins involved in cell adhesion, and to the actin cytoskeleton (47). Furthermore, β-catenin is also localized in the nucleus, binds the transcription factor LEF1/TCF (48) and its nuclear translocation is regulated by the cytoplasmic anchoring to cadherins (49). The similarity between Fe65-APP behaviour and that of β-catenin-cadherins is further supported by the observation that β-catenin lacks a classical NLS and is therefore imported in the nucleus by an unusual pathway (50). Another example of the extranuclear anchoring of transcription factors is given by Notch (51) and SREBP (52), whose precursors are membrane proteins which, following the proteolytic cleavage of their transmembrane helix, release in the cytosol a mature transcription factor that translocates to the nucleus. The case of Notch is particularly related to APP because of the involvement of presenilin dimers in the proteolytic cleavage of both proteins (51). This point deserves a particular attention and further experiments are needed to evaluate whether the APP processing by γ-secretase

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could result in the targeting of Fe65 to the nucleus, through the cleavage of the transmembrane domain of APP. The understanding of the functions of the complex protein-protein interaction network centred at the cytosolic domain of APP is an important step towards the discovery of APP functions and, possibly, of the molecular mechanisms regulating its amyloidogenic processing. The observations reported in this paper strengthen the hypothesis of a possible signalling mechanism linking APP to nuclear functions through Fe65 and its nuclear partner CP2/LSF/LBP1. This possibility is strongly supported by the recent finding that CP2/LSF/LBP1 gene, located at chromosome 12, is a genetic determinant of Alzheimer’s disease (53).

AcknowledgementsWe thank L. Nitsch, S. Bonatti, C. Garbi and C. Zurzolo for helpful discussion. This work was supported by grants from V Framework programme (contract n. QLK6-1999-02238) EU, Telethon (grant n. E.522), MURST-PRIN and CNR “Programma Biotecnologie MURST L.95/95”, Italy.

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Legends for Figures

Figure 1. Intracellular localization of Fe65. Exponentially growing COS7 cells were transfected with vectors driving the expression of GFP-Fe65 fusion proteins or of Fe65-HA. Panel A shows the western blot with anti-Fe65 antibody of total extracts from COS7 cells transiently transfected with CMV-Fe65 (lane a) that drives the expression of wild type Fe65, Fe65-GFP (lane b) that directs the expression of a fusion protein in which the GFP is at the C-terminus of Fe65, or with GFP-Fe65 (lane c), in which GFP is at the N-terminus of Fe65. Panels from B to E show the GFP-Fe65 (panels B and D) and the Fe65-GFP (panels C and E) fluorescent signals in transfected COS7 cells. Panels B and C show the fluorescent signal detected in fixed cells, while panels D and E show the signal observed in living unfixed cells. Panel F shows COS7 cells expressing Fe65-HA stained with an anti-HA antibody. A fluorescein conjugated antibody was used a secondary antibody. In panel G, nuclear (N), cytosol-membrane (CM) and total (T) extracts prepared from exponentially growing PC12 cells were analyzed by western blot with anti-Fe65 antibody: lane a, nuclear extract, lane b, cytosol-membrane extract, lane c, total extract.

Figure 2. GFP-Fe65 accumulates within COS7 cell nuclei.Confocal microscopy analysis of GFP-Fe65 fluorescent signal was performed as described under Materials and Methods. Panels from a to p show shows optical sections at 1-µm intervals from the base (panel a) to the top (panel p) of the cells.

Figure 3. Structure and subcellular localization of FE65 mutants.Panel A reports a summary of the GFP-mutantFe65 fusion proteins used. The length of each deletion is indicated in the name of the construct. The GFP-Fe65C655F construct encodes a GFP-Fe65 fusion protein in which cysteine 655 is changed into a phenylalanine. Panels from B to G show the GFP-mutant-Fe65 fluorescent signals in transiently transfected COS7 cells: panel B, GFP-Fe65∆666-711; panel C, GFP-Fe65∆1-287 in fixed cells; panel D, GFP-Fe65∆1-287 in living unfixed cells; panel E, GFP-Fe65∆1-253; panel F, GFP-GFP; panel G, GFP-GFP-Fe65/191-290. Panel H shows the western blot of cell extracts from COS7 transfected with the constructs indicated in panel A. The blot was stained with anti-GFP JL8 antibody.

Figure 4. APP co-expression prevents GFP-Fe65 accumulation within COS7 cell nuclei. COS7 cells were co-transfected with APP695 and with the GFP-Fe65 expression vector. APP was

fixed and stained with 6E10 antibody which recognizes the extracellular domain of the protein (panel A) or, in cells permeabilized with 0.1% TritonX100 after fixation, APP was stained with 369 antibody which recognizes the C-terminal cytosolic domain of APP (panel B). Texas red-conjugated secondary antibodies were used. Panels A’and B’ show the GFP-Fe65 fluorescence of the cells in the same fields of panels A and B, and panels A’’ and B’’ show the nuclear staining by Hoechst reagent.

Figure 5. APP doesn’t prevent the nuclear translocation of GFP-Fe65 mutant proteins unable to interact with APP.COS7 cells were co-transfected with APP695 and with two mutant GFP-Fe65 proteins (GFP-

Fe65∆538-711 and GFP-Fe65C655F) one lacking the PTB2 domain (panels A-A) and the other one bearing a point mutation (panels B-B), both preventing the formation of the Fe65-APP complex. Panels A and B show the cells stained with anti-APP antibodies 6E10 (panel A) and 369 (panel B); panels A’ and B’ show the GFP-Fe65 fluorescent signals of the same fields of panels A and B.Panel C shows the western blot with 6E10 antibody (anti-APP) of cell extracts immunoprecipitated

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with anti-GFP antibody (lanes a and b). In this experiments COS7 cells were co-transfected with expression vectors encoding APP695 and GFP-Fe65 (lanes a-a’) or APP695 and GFP-Fe65C655F

(lanes b-b’).

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Zambrano and Tommaso RussoGiuseppina Minopoli, Paola de Candia, Alessandro Bonetti, Raffaella Faraonio, Nicola

prevents Fe65 nuclear translocationThe beta-amyloid precursor protein APP functions as a cytosolic anchoring site that

published online November 20, 2000J. Biol. Chem. 

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