From the Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, I-80131 Naples, Italy
Received for publication, August 11, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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In this study we addressed the question of the
intracellular localization of Fe65, an adaptor protein interacting with
the The The elucidation of the normal functions of APP could be important in
understanding the molecular basis of Alzheimer's disease and in
approaching its prevention and/or treatment, but such
elucidation has been elusive up to now. One of the
characteristics of this protein currently investigated is the complex
network of protein-protein interactions that is centered 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, suggesting that these proteins function as
adaptor proteins bridging APP to specific molecular pathways.
Three of these adaptor proteins belong to the Fe65 protein family; Fe65
was 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 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 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 low density lipoprotein receptor-related protein
(27). On the other hand, particularly at the presynaptic level, X11
forms a trimeric complex in vivo with CASK and VELI,
the orthologues of Caenorhabditis elegans Lin2 and Lin7
proteins, respectively (37-39), and interacts with Munc18 (40) and a
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
identified up to now suggest multiple intracellular locations of this
adaptor protein, considering that Mena is mainly connected with the
actin cytoskeleton remodelling system (42) and that 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 coexpression of APP, whose cytodomain functions as a
cytosolic anchoring element.
Generation of GFP-Fe65 Expression Vectors--
The 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
The Fe65 point mutant C655F cDNA was obtained by using the
QuickChangeTM kit (Stratagene) according to the manufacturer's
procedure. For the mutagenesis, the pEGFP-C1-Fe65 expression vector was
used as a template, and the following pair of oligonucleotides were used as primers: forward 5, 5'-CTGTGCAGGCTGACATTCATGCTCCGCTAC and reverse 6, 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 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 obtained by cloning in the HindIII site of
pRC-CMV (Invitrogen), a polymerase chain reaction fragment obtained by
using the following primers: forward 6, 5'-CCCAAGCTTACTAAGGCCATGTCTGTTCCA and reverse HA,
5'-CCCAAGCTTTCAAGCGTAATCTGGAACATCATATGGGTATGGGGTCTGGGATCCTAGAC.
Cell Culture Conditions, Transfections, and Fluorescence
Microscopy--
Cells were grown at 37 °C in the presence of
5% CO2; COS-7 cells were cultured in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (HyClone) and 1% penicillin/streptomycin mixture
(HyClone). PC12 cells were cultured in RPMI (Life Technologies, Inc.)
supplemented with 10% horse serum (Life Technologies, Inc.), 5% fetal
bovine serum (HyClone), and 1% penicillin/streptomycin mixture
(HyClone). For the expression of the GFP-Fe65 fusion proteins or of
Fe65-HA, 1.5 × 106 cells were transfected by
electroporation at 250 microfarad and 220 V with 20 µg of each
construct. For cotransfection 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 h 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 Laboratories). Fe65-HA was stained with the anti-HA polyclonal antibody Y-11 (Santa
Cruz Biotechnology).
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 laid 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
Analysis--
For 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 × g at 4 °C, and the
protein concentration was determined by the Bio-Rad assay according to
the manufacturer's instructions. 10 µg of extract were analyzed by
SDS-polyacrylamide gel electrophoresis followed by immunoblotting as
described (22). For the immunoprecipitation experiment, the cell
extracts (2 mg) were incubated with the monoclonal antibody anti-GFP
JL8 (CLONTECH) according to the manufacturer's instructions. The immunocomplexes were collected with 30 µl/sample of
protein A/G plus-agarose (Santa Cruz Biotechnology), resolved on
SDS-polyacrylamide gel electrophoresis 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 × g for 15 min at 4 °C. Nuclear and
cytosol-membrane extracts were prepared as described (36).
Intracellular Localization of Fe65--
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 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 (Fig.
1A). 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 (Fig. 1,
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. Fig. 1 also shows that
there is no significant difference between the localization of the
fluorescent signal in fixed cells (B and C) and
in living unfixed cells (D and E). The confocal
microscope analysis confirmed that the fluorescence bound to Fe65 is
present within the nuclei (Fig. 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 HA
epitope, and stained with an anti-HA antibody (Fig. 1F). In addition, in this case the Fe65-HA signal is significantly represented both in the nucleus and in the cytosol.
To evaluate whether endogenous Fe65 is also present in the nucleus of
nontransfected cells, we examined PC12 cells, in which the levels of
the protein are significantly higher than 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 Fig. 1G, Fe65 is
clearly present in the nuclear extract (N), whereas its
amount in the cytosol-membrane (CM) fraction is severalfold higher than in the nuclear fraction.
The WW-containing Region Is Responsible for Fe65 Nuclear
Translocation--
The 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 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 Fig.
3A, were transfected in COS7
cells, and this resulted in the expression of fusion proteins of the
expected sizes (Fig. 3H). The amino acid sequence of rat
Fe65 contains only one region, from amino acids 684 to 705, with
clustered basic residues
(RRX5RRX9KXKR)
that resembles a nuclear localization signal (NLS) and that 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
acids 666 to 711 (Fig. 3A). Fig. 3B shows that
the cellular localization of this GFP-Fe65
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 being 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 acids
1 to 290 that we demonstrated to be necessary for its nuclear
translocation (Fig. 3, A and H). As shown in Fig. 3F, a protein consisting of two consecutive GFP units is
excluded from the nucleus, as expected from its molecular size, whereas the GFP-GFP-Fe65 protein carrying the region from amino acids 191 to
290 is translocated into the nucleus (Fig. 3G). 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 an NLS (44). Another possibility is
that Fe65 is transported to the nucleus by a cargo system including a
protein containing an NLS. An example of this "piggyback" mechanism is that of cyclin B1, which, lacking an NLS, is translocated into the
nucleus in a complex containing cyclin F, which possesses two
functional NLSs (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 because it is mainly localized in
the cytosol). The attempt to use the WW domain of Fe65 as bait to find
its partners by the two-hybrid screening system 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.
Fig. 3G shows that, unlike the wild type GFP-Fe65 protein
that is present both in the nucleus and in the cytosol (Fig. 1), GFP-GFP-Fe65191-290 is restricted to the nucleus. This suggests that a
nuclear export signal present in Fe65 has been removed from the
GFP-GFP-Fe65191-290 protein. One possible nuclear export signal
(LX3LX2VXV)
present in Fe65 from residues 180 to 189 and absent from the
GFP-GFP-Fe65191-290 protein cannot be considered, because it is also
absent from GFP-Fe65 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 Endoplasmic Reticulum-Golgi Area--
The protein-protein
interaction network centered at the cytosolic domain of APP could be
involved in transmembrane signaling 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. Therefore, we analyzed the
possible relationship between Fe65 intracellular trafficking and APP
expression. To address this point we cotransfected 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 Fig.
4, in nonpermeabilized cells 6E10 antibody allowed us to stain the cells transfected with
APP695 expression vector (A), and some of these
cells at the same time expressed GFP-Fe65. In these cotransfected cells
Fe65-bound fluorescence is excluded from the nucleus, whereas in the
cells expressing only GFP-Fe65 the fluorescence is localized in the
nucleus (Fig. 4A'). The same experiment was done in
permeabilized cells, by using the 369 antibody, which recognizes the
cytosolic domain of APP. In these conditions, in addition to the
already observed exclusion from the nucleus of GFP-Fe65 wild type in
the cells coexpressing APP, a clear colocalization of GFP-Fe65 wild
type and APP was seen at the plasma membrane, in the perinuclear
cisternae, and in the area of the Golgi network (Fig. 4, B
and B'). The cotransfection of the GFP-Fe65 vector with the
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 that encodes
a GFP protein fused to the deletion mutant of Fe65
(GFP-Fe65
The intracellular localization that we observed in COS7 cells
cotransfected 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 colocalized 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 also possible that in MDCK-695 cells a fraction of Fe65
is translocated to the nucleus, but that the amount of this fraction is
below the detection limit of the immunomicroscopy procedure used.
According to this possibility, in nontransfected PC12 cells the amount
of endogenous Fe65 present in the nuclear extract is much lower than
that present in the cytosol-membrane fraction (Fig. 1G).
The inhibition of Fe65 nuclear import by 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
Another example of the extranuclear anchoring of transcription factors
is given by Notch (51) and SREBP (52), whose precursors are membrane
proteins that, 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 particular attention, and further experiments are needed to
evaluate whether the APP processing by
The understanding of the functions of the complex protein-protein
interaction network centered at the cytosolic domain of APP is an
important step toward 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 signaling 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 the CP2/LSF/LBP1 gene, located at
chromosome 12, is a genetic determinant of Alzheimer's disease
(53).
-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 that,
on the other hand, a small fragment of 100 residues, including this WW
domain, contains enough structural information to target a reporter
protein (green fluorescent protein (GFP)-GFP) to the nucleus. To
evaluate whether the Fe65-APP interaction could affect Fe65
intracellular trafficking, COS7 cells were cotransfected 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,
whereas the nuclear targeting of GFP-Fe65 mutants, unable to interact
with APP, is unaffected by the coexpression of APP, thus suggesting
that the interaction with APP anchors Fe65 in the cytosol.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-amyloid precursor protein
(APP)1 is a widely expressed
integral membrane protein that is involved in the pathogenesis of
Alzheimer's diseases. In fact, the main constituent of the Alzheimer's disease senile plaques, the
-amyloid peptide, 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
-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 the endoplasmic reticulum-Golgi network (5,
6), where it undergoes cleavage by another proteolytic activity named
-secretase-BACE-1 (7-10), which generates a transmembrane
12-kDa fragment. This fragment is then cleaved by the
-secretase
activity, which leads to the formation of
-amyloid
peptide. Recent evidence strongly suggests the identity of
-secretase and presenilin dimer (11, 12).
XNPXY motif (where
indicates a hydrophobic residue, and X indicates 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
XNPXY 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
-amyloid peptide in cultured cells (28, 29), whereas
X11-APP coexpression inhibits the proteolytic processing of APP (25, 30, 31). Lastly, APP seems to have other partners also, 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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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, forward 1, 5'-GAGCTCAAGCTTCTACTAAGCCATGTCTGTTC and reverse 1, 5'-GACCGCGGGCCCGCGGGGTCTGGGATCCTAG; for GFP-Fe65
666-711, forward 1 and reverse 2, 5'-GCCATCGGGCCCAAGCATCCAGACACTTCTGGTA; for
GFP-Fe65
538-711, forward 1 and reverse 3, 5'-GCCATCGGGCCCAAGCATTGCGCCTTCAGACA; for GFP-Fe65
1-287, forward 2, 5'-GAGCTCAAGCTTCCCCATCACAGGGGAACAG and reverse 1; for
GFP-Fe65
1-253, forward 3, 5'-GAGCTCAAGCTTCCGATCTACCGGCTGGATG and
reverse 1; for GFP-GFP-Fe65191-290, forward 3, 5'-ATATGAATTCTGGACCCCAGGYCCTCACAGATGG and reverse 4, 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: forward 4, 5'-ATATCTCGAGAGCTGGACGGCGACGTAAACG and reverse 5, 5'-ATAAGAATTCAGGTAGTGGTTGTCGGGCAGC. The GFP-GFP-Fe65191-290 was
obtained by cloning in frame the corresponding cDNA fragment downstream of the region encoding the second GFP in the GFP-GFP vector.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 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. 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 (w.t.) Fe65, with
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.
B-E show the GFP-Fe65 (B and D) and
the Fe65-GFP (C and E) fluorescent signals in
transfected COS7 cells. B and C show the
fluorescent signal detected in fixed cells, whereas D and
E show the signal observed in living unfixed cells.
F shows COS7 cells expressing Fe65-HA stained with an
anti-HA antibody. A fluorescein-conjugated antibody was used as
a secondary antibody. In 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.
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Fig. 2.
GFP-Fe65 accumulates within COS7 cell
nuclei. Confocal microscopy analysis of GFP-Fe65 fluorescent
signal was performed as described under "Experimental Procedures."
a-p show optical sections at 1-µm intervals from the base
(a) to the top (p) of the cells.
666-711 is identical to
that of the GFP-Fe65 wild type protein, 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, which is
completely excluded from the nucleus (Fig. 3C). The same
exclusion from the nucleus was also observed in living unfixed cells
(Fig. 3D). To further restrict the sequence involved in the
Fe65 nuclear localization, we generated some more deletion mutants. As
shown in Fig. 3E, one of these mutants,
GFP-Fe65
1-253 (Fig. 3A), 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 resides from amino acid 250 to amino acid
290, a region that 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, whereas deletion mutants of Fe65, lacking the
N-terminal region, are only found in the cytosolic extracts (36).
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Fig. 3.
Structure and subcellular localization of
Fe65 mutants. A reports a summary of the GFP-mutant Fe65
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.
B-G show the GFP-mutant Fe65 fluorescent signals in
transiently transfected COS7 cells: B, GFP-Fe65 666-711;
C, GFP-Fe65
1-287 in fixed cells; D,
GFP-Fe65
1-287 in living unfixed cells; E, GFP-Fe65
1-253; F, GFP-GFP; G, GFP-GFP-Fe65191-290.
H shows the Western blot of cell extracts from COS7 cells
transfected with the constructs indicated in A. The blot was
stained with anti-GFP JL8 antibody.
1-253, which is located both in the nucleus and
in the cytosol (Fig. 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 interaction with an
exported protein. CP2/LSF/LBP1 interacts with the PTB1 domain of Fe65
and 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 nuclear
export signal.
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Fig. 4.
APP coexpression prevents GFP-Fe65
accumulation within COS7 cell nuclei. COS7 cells were
cotransfected 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 (A), or in cells
permeabilized with 0.1% Triton X-100 after fixation, APP was stained
with 369 antibody, which recognizes the C-terminal cytosolic domain of
APP (B). Texas red-conjugated secondary antibodies were
used. A'and B' show the GFP-Fe65 fluorescence of
the cells in the same fields as A and B, and
A" and B" show the nuclear staining by Hoechst
reagent.
538-711; see Fig. 3A) that lacks the PTB2
domain that is responsible for binding to APP. Fig.
5, A and A' show that the GFP-Fe65
538-711 mutant is located in the nucleus and that
in the cells coexpressing APP695 the nuclear translocation of the Fe65 mutant is unaffected. Furthermore, this GFP-Fe65
538-711 mutant does not accumulate in the perinuclear cisternae or in the Golgi
area. An identical behavior was observed when the cells were
transfected with a GFP-Fe65 construct bearing a point mutation changing
the Cys-655 of Fe65 into a Phe (Fig. 5, B and
B'). This Fe65 mutant is unable to interact with APP (20),
as demonstrated by the coimmunoprecipitation experiment reported in
Fig. 5C. In fact, APP was not coimmunoprecipitated with Fe65
in extracts from COS7 cells transfected with Fe65C655F and
APP695, whereas APP coimmunoprecipitates with wild type
Fe65.
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Fig. 5.
APP does not prevent the nuclear
translocation of GFP-Fe65 mutant proteins unable to interact with
APP. COS7 cells were cotransfected with APP695 and
with two mutant GFP-Fe65 proteins (GFP-Fe65 538-711 and
GFP-Fe65C655F), one lacking the PTB2 domain (A-A') and the
other one bearing a point mutation (B-B'), both preventing
the formation of the Fe65-APP complex. A and B
show the cells stained with anti-APP antibodies 6E10 (A) and
369 (B); A' and B' show the GFP-Fe65
fluorescent signals of the same fields as A and
B. C shows the Western blot with 6E10 antibody
(anti-APP) of cell extracts immunoprecipitated with anti-GFP antibody
(lanes a and b). In this experiment COS7 cells
were cotransfected with expression vectors encoding APP695
and GFP-Fe65 (lanes a-a') or APP695 and
GFP-Fe65C655F (lanes b-b'). I.P.,
immunoprecipitation; w.b., Western blot.
-catenin. This protein plays an important role in the
Wingless-signaling pathway (46); it functions as an adaptor protein binding to cadherins, membrane proteins involved in cell adhesion, and to the actin cytoskeleton (47). Furthermore,
-catenin is also localized in the nucleus and binds the transcription factor LEF1/TCF (48), and its nuclear translocation is regulated by cytoplasmic anchoring to cadherins (49). The similarity between Fe65-APP behavior and that of
-catenin-cadherins is further
supported by the observation that
-catenin lacks a classical NLS and
is therefore imported in the nucleus by an unusual pathway (50).
-secretase could result in
the targeting of Fe65 to the nucleus through the cleavage of the
transmembrane domain of APP.
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ACKNOWLEDGEMENTS |
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We thank L. Nitsch, S. Bonatti, C. Garbi, and C. Zurzolo for helpful discussion.
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FOOTNOTES |
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* This work was supported by grants from V Framework program (Contract Number QLK6-1999-02238) EU, Telethon (Grant Number E.522), MURST-PRIN, and Consiglio Nazionale delle Ricerche "Programma Biotecnologie MURST L.95/95," Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dipartimento di
Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, I-80131 Naples, Italy. Tel.: 390817463131; Fax:
390817464359; E-mail: russot@dbbm.unina.it.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M007340200
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ABBREVIATIONS |
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The abbreviations used are:
APP, -amyloid precursor protein;
GFP, green fluorescent protein;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
NLS, nuclear
localization signal;
PTB, phosphotyrosine binding domain;
EGFP, enhanced green fluorescent protein.
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