From the Dipartimento di Biochimica e
Biotecnologie Mediche, Università degli Studi di Napoli Federico
II, Napoli 80131, Italy,
CEINGE
biotecnologie avanzate, Napoli 80131, Italy
Neurodegenerative
Disease Group, Aventis Pharma, Vitry-sur-Seine 94400, France, and
the ** Proteomics and Mass Spectrometry Laboratory, ISPAAM,
National Research Council, Napoli 80147, Italy
Received for publication, November 21, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The The functions of APP and its proteolytic processing are still
unknown. However, although the functions of the APP ectodomain remain
elusive, there is increasing evidence that its cytodomain is the center
of a complex network of interactions with several proteins, involved in
vesicle transport and in signal transduction. In fact, it was
demonstrated that APP cytoplasmic domain interacts with kinesin light
chain and contributes to vesicles transport (4), thus suggesting that
APP cleavage could regulate the transport of vesicles in the axons. On
the other hand, the APP cytodomain binds several PTB domain-containing
proteins, some of which are involved as adaptor proteins in signal
transduction. Fe65 is the first protein that was found to form a stable
complex with the cytosolic domain of APP, through one of the two PTB
domains it possesses (5, 6). Fe65 is an adaptor protein that
interacts with APP, Mena (7) (the mammalian orthologue of the
product of the enabled gene of Drosophila),
tyrosine kinase Abl (8) (through its WW domain), transcription factor
LSF (9), the histone acetyltransferase Tip60 (10), and low density
lipoprotein-receptor-related protein LRP (11) (through its second PTB
domain). The involvement of Mena and Abl in the complexes, including
APP and Fe65, is in agreement with the finding that APP cytodomain also
interacts with mDab1 (12), the orthologue of the Drosophila
disabled gene, and with the Abl TK, through its SH2 domain
(8), given that genetic manipulation of the fly indicated that
enabled, disabled, and DAbl are associated
in the same pathway (13). Another interactor of APP is X11 (14), an
adaptor protein that forms complexes with various proteins, including
Munc18 and CASK (15). More recently, two adaptors involved in signal
transduction, Shc and Jip1, have been demonstrated to bind to APP and
AID through their PTB domains (16-19).
The evident complexity of this protein-protein interaction network
suggests that APP could be a multifunctional molecule that anchors
several different oligomeric complexes close to the membrane, possibly
in specific subdomains such as caveolae (20), and/or that APP regulates
the availability of these complexes in their final destination, upon
APP cleavage and detachment of the APP cytodomain from the membrane.
A crucial point that should be addressed to further study the
possible interplay of APP and transduction pathways involving the above
mentioned proteins concerns the molecular mechanisms that induce APP
processing. In this report we show that PDGF-BB is a potent activator
of APP Generation of HeLaAG Clones--
A vector driving the expression
of a fusion protein, consisting of the human APP695
followed by a flexible hinge of ten glycines and by the entire yeast
transcription factor Gal4, has been generated by cloning into RcCMV
vector (Invitrogen) cDNA fragments amplified using as template the
human APP695 cDNA and the yeast Gal4 cDNA. The
following specific oligonucleotide primers (CEINGE) were used for PCR amplifications (94 °C, 1 min; 64 °C, 1 min;
72 °C, 7 min; for 40 cycles): forward hAPP695
HindIII
(5'-CCCAAGCTTACTAAGGCCATGCTGCCCGGTTTGGCACTGC-3') and
reverse hAPP695 Apa/NotI
(5'-CATCGGGCCCCTACGCGGCCGCGTTCTGCATCTGCTCAAAGAACTTG-3'); forward yGAL4 Not/10Gly
(5'-AAGGAAAAAAGCGGCCGCTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTAAGCTACTGTCTTCTATCGAACAAGC-3') and reverse yGAL4 ApaI
(5'-CATCGGGCCCTTACTCTTTTTTTGGGTTTGGTGGGG-3'). Restriction sites are underlined, and the ten glycine codons are in italic. This APP-Gal4 expressing vector, containing the
neomycin resistance gene, has been transfected into HeLa cells by
calcium-phosphate method, and after a 14-day G418 selection (900 µg/ml final concentration), several G418-resistant clones have been
isolated. Two pools of these clones have been used (HeLaAG).
The cleavage of APP-Gal4 fusion protein has been assayed by transiently
transfecting HeLaAG cells (5 × 105 cells/60-mm
dishes) by calcium-phosphate method with G5BCAT vector (3 µg), in
which the transcription of chloramphenicol acetyltransferase (CAT) gene
is under the control of a Gal4-dependent promoter (21). CAT
expression was measured by using colorimetric CAT enzyme-linked immunosorbent assay (Roche Molecular Biochemicals). Other transfections of HeLaAG cells were carried out by using the calcium-phosphate method;
all the plasmids were used at 3 µg each, and the total amount of DNA
in co-transfections was always brought to 10 µg with RcCMV vector.
Cell Culture Conditions--
Wild type HeLa and HeLa AG cells
were grown at 37 °C in the presence of 5% CO2 in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin (all from HyClone). C6 and NIH3T3 cells and co-cultures
were at 37 °C in the presence of 5% CO2 in RPMI medium
(Invitrogen) supplemented with 10% fetal bovine serum and antibiotics.
Wild type HeLa or HeLa AG cells, 24 h after transfection with
G5BCAT vector, were treated for the indicated times with 40 ng/ml
recombinant human PDGF-BB (Sigma), 5 or 10 µg/ml protein fraction
precipitated with 40% AS or 200-µl fractions eluted from Sephadex
G-75, diluted in DMEM without serum to a final volume of 2 ml.
Inhibitors were added at the indicated times at the following concentrations: 10 µM PP2 (Calbiochem), 2 µM AG1296 (Calbiochem), 30 µM genistein
(Calbiochem), 10 µM
HEK293 and CHO cells were grown in the same conditions as HeLaAG.
HEK293 were transfected with 0.5 µg of human APP695
expression vector and 0.5 µg of SrcYF vector by LipofectAMINE 2000 (Invitrogen) in 35-mm plates. Total A Purification of the Activity That Induces APP-Gal4
Cleavage--
C6 cells were grown as described above in 100 dishes of
150-mm diameter (Falcon) to confluence; cell sheets in each dish have been washed twice with phosphate-buffered saline (PBS), and then cells
were cultured in RPMI medium without serum. After 3 days of incubation,
3 liters of conditioned medium was harvested, centrifuged at 1000 rpm
for 20 min to remove debris, and concentrated to 480 ml by using a
Centriplus YM-3000 (Amicon). Then, 104 g of ammonium sulfate was
added to the concentrated conditioned medium to obtain a 40% ammonium
sulfate solution, which was stirred overnight at 4 °C and then
centrifuged at 9000 rpm for 2 h. 70% AS saturation was
reached by adding 87.4 g of ammonium sulfate to the 40% saturated solution, and 100% AS saturation was reached by adding 100.3 g of
ammonium sulfate to the 70% saturated solution.
The precipitates were dissolved in 15 ml of PBS and dialyzed against
5 × 2-liter changes of PBS. 1 mg of this sample was separated by
FPLC onto a Sephadex G-75 column (30 g of swollen resin,
pre-equilibrated in PBS) and run in this solvent at 0.25 ml/min
while collecting 400-µl fractions every 1.6 min.
Bands from SDS-PAGE were excised from the gel, triturated, and washed
with water. Proteins were reduced in-gel, S-alkylated with
iodoacetamide, and digested with trypsin as previously reported (22).
Digested aliquots were subjected to a desalting/concentration step on
µZipTipC18 (Millipore Corp., Bedford, MA) before MALDI-TOF mass
spectrometry analysis. Peptide mixtures were loaded on the instrument
target, using the dried droplet technique and
Immunodepletion of 40% AS fraction was obtained by incubating 20 µg
of the fraction diluted in 500 µl of PBS with 60 µg of anti-PDGF
antibody (Sigma) or with 60 µg of mouse IgG (Sigma) for 2 h at
4 °C. Then the mixtures were chromatographed on 20 µl of Protein
AG-Sepharose (Santa Cruz Biotechnology) for 30 min at 4 °C, and,
after centrifugation, the supernatants were diluted in DMEM without
serum to a final volume of 2 ml.
Preparation of Cell Extracts and Western Blotting
Analyses--
For CAT assay, transiently transfected HeLa AG cells
were harvested in cold TEN (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl), frozen at
For Western blotting analyses, HeLa AG cells were harvested in
cold PBS, resuspended in lysis buffer (40 mM Tris-HCl, pH
7.2, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA,
0.2 mM phenylmethylsulfonyl fluoride, 100 µg/ml
aprotinin, 100 µg/ml leupeptin) and kept in ice for 15 min. Then
total extracts were clarified by centrifugation at 14,000 rpm at
4 °C. 20 µg of each extract or 20 µl of pooled fractions eluted
from the G-75 column was electrophoresed on 4-12% SDS-polyacrylamide
gradient gel under reducing conditions and transferred to Immobilon-P
membranes (Millipore). Filters were then blocked in 5% nonfat dry milk
in T-PBS solution (PBS and 0.05% Tween) and incubated with appropriate
dilutions of primary antibody, overnight at 4 °C. The excess
antibody was removed by sequential washing of the membranes in T-PBS,
and then a 1:5000 dilution of the appropriate secondary antibody
(horseradish peroxidase-conjugated) was added to filters for 30 min, at
room temperature. The excess was removed by sequential washing of the
membranes in T-PBS, and the signals were detected by chemiluminescence,
using the ECL system (Amersham Biosciences). The antibodies used and
their dilutions were: anti-PDGF (Sigma), 1:750; anti-Gal4DBD
(Calbiochem), 1:1000; anti-APP 6E10 (Sigma), 1:1000; anti-APP CT695
(Zymed Laboratories Inc.), 1:250; anti-phosphoERK
(Santa Cruz Biotechnology), 1:1000; and anti-phosphoAkt (Santa Cruz),
1:1000.
C6 Cell-conditioned Medium Induces Gal4-dependent-CAT
Gene Transcription in HeLa Cells Expressing APP-Gal4 Fusion
Protein--
We examined the possibility that extracellular signals
could induce the
To evaluate both cell-anchored and secreted factors that
could activate APP-Gal4 proteolytic processing, the first experimental approach we used consisted of 1) a co-culture of HeLaAG cells, transiently transfected with G5BCAT plasmid, with various cell lines of
different origin and 2) an assay of CAT accumulation in HeLaAG cultures
pure or co-cultured with these cells. Fig. 1C shows that
co-culturing of HeLaAG with C6 cells resulted in a significant increase
of the CAT expressed by HeLaAG, whereas no change was observed in the
co-cultures with other cell lines, such as NIH3T3 fibroblasts. C6 cells
are derived from a rat glioma and are known to secrete several growth
factors (24). Therefore, we examined whether the conditioned medium
from C6 cells mimics the effect of CAT accumulation observed in the
co-cultures. As shown in Fig. 1C, HeLaAG cells, grown in the
presence of C6-conditioned medium, express higher levels of CAT
compared with the cells grown in the conditioned medium from HeLa cells
or from NIH3T3 cultures.
Purification of the APP-Gal4 Cleavage-inducing Activity--
A
large-scale preparation of C6-conditioned medium was used as a source
for the purification of the one or more molecules that induce the CAT
accumulation in HeLaAG cells transfected with G5BCAT vector. Fig.
2 shows the steps of this purification
based on ammonium sulfate (AS) precipitation, size-exclusion
chromatography, and SDS-PAGE. The activity is restricted to the 40% AS
fraction (Fig. 2A), which was applied on FPLC equipped with
a Sephadex G-75. Eluted fractions from the chromatography were assayed
for their ability to induce CAT accumulation in HeLaAG cells, and the
results allowed us to identify two peaks of activity of about 70 and 30 kDa, respectively (Fig. 2B). SDS-PAGE of the proteins present in the relevant fractions, compared with fractions devoid of
activity, suggested that one band of about 15 kDa could be a good
candidate (see Fig. 2C). This band, separately excised from
the lanes of the corresponding active fractions, was digested with
trypsin and analyzed by MALDI-TOF mass spectrometry. Peptide mass
fingerprint analysis and non-redundant sequence data base matching in
both cases allowed its unambiguous identification as PDGF-B. To
confirm the identification, the relevant fractions were electrophoresed
and blotted with a PDGF antibody. This blot demonstrated that PDGF is
present only in the fractions that activate the accumulation of CAT in
HeLaAG cells (Fig. 2D).
PDGF Induces a
To rule out the possibility that PDGF treatment induces CAT
accumulation through a mechanism independent from the cleavage of
APP-Gal4, we exposed wild type HeLa cells mock transfected or
transfected with Gal4 to 40 ng/ml PDGF-BB. As shown in Fig. 4A, PDGF-BB treatment did not
modify the accumulation of CAT in these experimental conditions.
Therefore, the increase of CAT concentration observed in HeLaAG cells
exposed to PDGF could be due to an activation of the CAT gene
transcription by GAL4 released upon the cleavage of APP-Gal4. To
address this point, extracts from HeLaAG cells exposed to PDGF or AS
40% fraction were analyzed by Western blot with anti-APP or anti-Gal4
antibodies. These experiments showed the presence, in extracts from
HeLaAG cells exposed to PDGF or 40% AS fraction, of a band of a size
very similar to that of Gal4, with both the Gal4 antibody and the CT695
antibody, recognizing the APP C-terminal domain (see Fig.
4B). A similar blot was challenged with the 6E10 antibody,
which was directed against the N-terminal sequence of the
To evaluate whether the observed cleavage of APP-Gal4 requires the
PDGF-induced APP-Gal4 Cleavage Functions through an
Src-dependent Pathway--
There are many pathways
activated following PDGF-R interaction with its cognate growth factor.
Tyrosine-phosphorylated PDGF-R activates the Ras-MAPK pathway through
Grb2/SOS and Shc/Grb2/SOS. The possible involvement of this pathway in
the APP-Gal4 processing was explored by treating HeLaAG cells exposed
to PDGF with the inhibitor of ERKs, PD098059; this inhibitor does not
modify the effects of both PDGF and 40% AS fraction (Fig.
5). Another pathway that mediates the
effects of PDGF-R activation is that of PI3K-Akt. Also, Fig. 5
shows that the PI3K inhibitor wortmannin does not affect the CAT
accumulation induced by both PDGF and 40% AS fraction.
Src and other members of the Src non-receptor TK family interact with
and are activated by PDGF-R (26). To explore this pathway, HeLaAG cells
have been treated with a specific inhibitor of Src TK, PP2, and with a
related compound unable to inhibit Src (PP3). The treatment with PP2 of
HeLaAG cells almost completely abolished the accumulation of CAT
observed upon the exposure to either PDGF or 40% ammonium sulfate
fraction, whereas the treatment with PP3 was completely ineffective
(see Fig. 5).
To further explore this finding, HeLaAG cells were transiently
transfected with SrcYF vector expressing a constitutively active Src
mutant (27). As shown in Fig.
6A, the expression of active Src resulted in the accumulation of CAT in the absence of stimulation by either PDGF or purified fractions. Accordingly, HeLaAG cells transfected with a dominant negative mutant of Src (SrcYFKM) (28) and
exposed to PDGF or to 40% AS fraction showed a significantly decreased
accumulation of CAT, compared with mock transfected cells exposed to
PDGF.
The possible effectors downstream of Src are not completely understood.
One of these downstream factors is the non-receptor TK Abl (29). The
possible role of Abl TK in APP-Gal4 cleavage was explored by
transfecting HeLaAG cells with a constitutively active mutant of Abl
(Abl-PP) (30). Under these conditions no induction of APP-Gal4 cleavage
was observed, thus indicating that this kinase is not involved in this
phenomenon. On the contrary, another molecule that has been recently
observed to be activated by PDGF and Src is Rac1, which belongs to the
family of Rho G-proteins. The transfection of HeLaAG cells with a
vector driving the expression of a constitutively active form of Rac
(RacQL) (31) resulted in an increase of CAT comparable to that observed
upon the transfection with SrcYF, and the co-transfection of SrcYF with
a dominant negative mutant of Rac (RacN17) strongly decreased the
amount of CAT compared with that accumulated in the cells transfected
only with SrcYF (see Fig. 6A). Furthermore, a similar
inhibition of CAT accumulation, following the exposure to either PDGF
or 40% ammonium sulfate fraction, was observed in the cells
transfected with RacN17.
To ascertain whether Src and Rac1, like PDGF, also activate APP-Gal4
processing through a PDGF Induces the Generation of A The proteolytic processing of APP leading to the generation of
A Most of the available data on the regulation of APP processing concerns
with sAPP secretion (for a review see Ref. 32). It is well demonstrated
that the activation of muscarinic receptor induces an increased
secretion of sAPP (33), and a similar phenomenon has been reported also
for metabotropic glutamate receptor (34) and for serotonin receptors
(35). These effects are regulated through a PKC-dependent
pathway (33), and, accordingly, it is well known that activated PKC
induces sAPP secretion and inhibits A The relevance of the reported results for neuronal APP functions and
for the pathogenesis of AD should be addressed through further work.
However, PDGF-R, Src, and Rac, although widely expressed in many
different cell types, are known to play significant roles in the
nervous system. In fact, it was clearly documented that PDGF
The most known effectors of Rac1 are the PAK serine/threonine kinases,
which are activated through the binding of Rac-GTP or Cdc42-GTP to
their N-terminal autoinhibitory domain (for a review see Ref. 49). A
second known effector downstream of Rac1 is the kinase Cdk5 (50), and
the observation that APP The results reported here support the hypothesis that other
extracellular signals, different from PDGF and known to induce Src
and/or Rac1, could trigger the processing of APP. In fact, numerous
signaling pathways converge on Src: (i) several other tyrosine kinase
receptors, such as nerve growth factor receptor, epidermal growth
factor receptor, and fibroblast growth factor receptor, are able to
activate Src (52-54); (ii) Src is activated by engagement of integrins
during cell interaction with extracellular matrix (55); (iii)
G-protein-coupled receptors activate Src, as in the case of thrombin
(56, 57); and (iv) voltage-dependent and ligand-gated
channels have been demonstrated to interact with Src such as, for
example, the N-methyl-D-aspartic acid channel (58).
Among the possible targets of the pathway described above are
secretases and APP. The structure of There are several results indicating that the processing of APP
by Taken together these data suggest the possibility that the activation
of PDGF-R, Src, and Rac1 could be relevant for the generation of A-amyloid peptide (A
) present in
the senile plaques of Alzheimer's disease derives from the cleavage of
a membrane protein, named APP, driven by two enzymes, known as
- and
-secretases. The mechanisms regulating this cleavage are not
understood. We have developed an experimental system to identify
possible extracellular signals able to trigger the cleavage of an
APP-Gal4 fusion protein, which is detected by measuring the expression
of the CAT gene transcribed under the control of the Gal4 transcription
factor, which is released from the membrane upon the cleavage of
APP-Gal4. By using this assay, we purified a protein contained in the
C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the
-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the
activation of a pathway downstream of the PDGF-receptor, which includes
the non-receptor tyrosine kinase Src and the small G-protein Rac1.
These findings are confirmed by the observation that a constitutively
active form of Src increases A
generation and that, in cells stably
expressing APP, the generation of A
is strongly decreased by the Src
tyrosine kinase inhibitor PP2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Amyloid (A
)1
deposition in the so-called amyloid plaques is one of the main
features of Alzheimer's pathology.
-Amyloid consists of ~4-kDa
peptides derived from the proteolytic processing of a membrane protein
named amyloid precursor protein (APP). This amyloidogenic processing is
driven by two enzyme activities,
-site APP cleaving enzyme (BACE)
and
-secretase. BACE cleaves APP at 28 residues from the boundary
between the extracellular/intraluminal domain of APP and the
transmembrane domain of the protein (for a review see Ref. 1),
releasing a large soluble protein, including nearly all the
extracellular/intraluminal part of APP, and a short transmembrane peptide, including the 99 C-terminal residues of APP.
This transmembrane C99 stub is a substrate for the
-secretase activity, which cleaves it, in a presenilin-dependent
fashion, within the membrane
-helix, giving rise to the A
peptide
40-42 amino acids long and to a peptide named APP intracellular domain (AID), which includes the small C-terminal cytosolic domain of APP (for
a review see Refs. 2 and 3).
-
cleavage, giving rise to an increased generation of A
through a pathway involving the non-receptor tyrosine kinase Src and
the small G-protein Rac1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-secretase inhibitor compound X
(Calbiochem), 100 µM PD098059 (Sigma), and 100 nM Wortmannin (Sigma).
peptide was measured by
sandwich enzyme-linked immunosorbent assay with 6E10 and 4G8 antibodies.
-cyano-4-hydroxycinnamic as matrix, and analyzed by using a
Voyager-DE PRO mass spectrometer (Applied Biosystems, Framingham, MA).
The PROWL software package was used to identify proteins unambiguously
from an independent non-redundant sequence data base (23).
80 °C for
30 min, and resuspended in lysis buffer (10 mM Hepes, pH
7.9, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5%
glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 400 mM NaCl). Total extracts were clarified by centrifugation
at 14,000 rpm at 4 °C, and protein concentration was determined by
Bio-Rad assay; for CAT concentration measurement, 150 µg of each
protein extract was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-
-secretase-mediated cleavage of APP. To address this point we developed an experimental system based on a recombinant protein in which the yeast Gal4 transcription factor is fused to the
cytosolic C-terminal end of APP695. This system is based on
the prediction that, in cells expressing APP-Gal4, upon the cleavage of
this molecule by
-
-secretase activities, AID-Gal4 is released
from the membrane and should become available to activate the
transcription of the chloramphenicol acetyltransferase (CAT) gene cloned under the control of five Gal4 cis-elements in the G5BCAT
vector (21) (see Fig. 1A).
Based on this experimental design, HeLa cells were transfected with a
vector driving the expression of APP-Gal4 fusion protein and G418
resistance gene, and several clones stably expressing APP-Gal4 have
been isolated. Fig. 1B shows a Western blot of the
extracts from several HeLa clones challenged with either APP or Gal4
antibodies and demonstrates the expression of a protein recognized by
both antibodies. The experiments reported below were conducted by using
two pools of these clones, HeLaAG1-8 and HeLaAG9-14, thereafter
indicated as HeLaAG.
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Fig. 1.
Isolation and characterization of HeLa cell
clones stably expressing APP-Gal4 fusion protein. A,
schematic representation of the experimental system: APP-Gal4 fusion
protein is cleaved by - and
-secretases, and this results in the
release from the membrane of Gal4 protein fused to the intracellular
domain of APP (AID-Gal4). This protein then activates the transcription
of the CAT gene cloned under the control of five Gal4 cis-elements.
B, Western blot analyses of lysates from HeLa cells
transfected with human APP695 or with Gal4 expression
vectors and from two pools of HeLa clones (HeLaAG1-8, HeLaAG9-14)
stably expressing APP-Gal4 fusion protein. The two Western blots are
with APP 6E10 antibody, recognizing the extracellular domain of APP,
and with Gal4 antibody, respectively. One asterisk indicates
wild type APP bands, two asterisks indicate APP-Gal4 bands,
and the arrowhead indicates wild type Gal4 bands. C, HeLaAG
cells transfected with G5BCAT vector were co-cultured with either
NIH3T3 or C6 cells or cultured in the presence of the conditioned
medium of these cell lines. For co-cultures, cells were plated at the
indicated cell numbers, harvested 72 h after plating and their
extracts were assayed for CAT concentration. Conditioned media from
72 h cultures of the indicated cells were added to 2.5 × 105 HeLaAG cells. Extracts from cells harvested 48 h
after the exposure to conditioned medium were assayed for CAT
concentration. Standard deviations of triplicate experiments are
reported.
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Fig. 2.
Purification of the activity present in the
C6-conditioned medium, which induces CAT expression in HeLaAG
cells. A, 3 liters of C6-conditioned medium was concentrated and
fractionated by ammonium sulfate precipitation. The fractions obtained
with a salt saturation of 40, 70, or 100% were dialyzed and assayed
for their ability to induce CAT accumulation in HeLaAG cells
transfected with G5BCAT vector. The concentrations of assayed samples
were: 40% AS, 5 µg/ml; 70% AS, 200 µg/ml; 100% AS, 200 µg/ml.
Standard deviations of triplicate experiments are reported.
B, 40% AS fraction was applied on a Sephadex G-75 column
and chromatographed by FPLC. Fractions of 400 µl were collected and
assayed for their ability to induce CAT accumulation in HeLaAG cells.
The calibration of the column with pure standards of known molecular
mass indicated that the two peaks of activity were eluted as ~70 and
30 kDa proteins, respectively. C, fractions of the Sephadex
chromatography containing (7 and 23) and not
containing (9, 11, and 20) the
activity were concentrated and electrophoresed on a reducing SDS-PAGE
and silver-stained. The arrow indicates a band of about 15 kDa present in fractions containing the activity. D, pooled
fractions containing (7-8 and 23-24) or not containing (3-4, 10-11,
14-16, 17-18, 19-20, 21-22, 25-27) the activity were
electrophoresed on a reducing SDS-PAGE and blotted with a PDGF
antibody, thus confirming the identification made by MALDI-TOF mass
spectrometry.
-Secretase-dependent Cleavage
of APP-Gal4 Protein--
HeLaAG were exposed to 40 ng/ml of purified
human PDGF-BB for 12 h, and this resulted in a dramatic induction
of CAT expression (Fig. 3). This
phenomenon depends on the activation of the PDGF receptor (PDGF-R),
considering that the treatment of HeLaAG cells with a TK-nonspecific
inhibitor such as genistein or with PDGF-R inhibitor AG1296 resulted in
a significant decrease of CAT accumulation, following the treatment
with PDGF. Although, unexpectedly, PDGF is present also in the
~70-kDa fraction of the size exclusion chromatography (see Fig. 2,
C and D), it cannot be excluded that other
molecules with an activity similar to that of PDGF could be also
present in the C6-conditioned medium. To address this point, HeLaAG
cells were treated with the 40% ammonium sulfate fraction and with the
PDGF-R inhibitor. Also in this case, the accumulation of CAT was
prevented by the PDGF-R inhibitor (see Fig. 3), thus strongly
supporting that PDGF is the only factor, present in C6-conditioned
medium, that activates APP-Gal4 cleavage. Furthermore, immunodepletion
of the 40% AS fraction with anti-PDGF antibody resulted in the
abolishment of the CAT accumulation observed upon exposure of HeLaAG
cells to pure 40% AS fraction, whereas the depletion with mouse IgG
was completely ineffective (see Fig. 3). Therefore, the activity
present in the 70-kDa fraction could be a multimer of the PDGF-B
subunit.
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Fig. 3.
PDGF-BB contained in C6 cell-conditioned
medium induces CAT accumulation in HeLaAG cells. HeLaAG cells
transfected with G5BCAT vector were exposed to 40 ng/ml recombinant
PDGF-BB or to 10 µg/ml 40% AS fraction for 24 h before
harvesting. In the same conditions the cells were also exposed, as
indicated, to 30 µM genistein or to 2 µM
AG1296, which are a general TK inhibitor and a PDGF-R TK inhibitor,
respectively. To ascertain whether the 40% AS fraction also contains
factors, other than PDGF-BB, activating CAT expression, the 40% AS
fraction was immunodepleted either with anti-PDGF antibody ( -PDGF)
or with mouse IgG (mIgG), and HeLaAG cells were exposed to these
mixtures (ID 40%AS). Standard deviations of triplicate experiments are
reported.
-amyloid
peptide. This antibody recognizes the uncleaved APP-Gal4 but not the
cleaved molecule. This indicates that the cleaved molecule contains the
C-terminal domain of APP (AID-Gal4) and not the N-terminal sequence of
A
.
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Fig. 4.
PDGF-induced CAT accumulations depends on
APP-Gal4 cleavage by -secretase activity.
A, wild type HeLa cells were transfected with G5BCAT vector
alone (mock) or with both G5BCAT vector and Gal4 expression vector
(Gal4) and treated with 40 ng/ml recombinant PDGF-BB or with 10 µg/ml
of the 40% AS fraction for 24 h before harvesting. The amount of
CAT was measured in triplicate experiments, and standard deviations are
reported. B, cell extracts from HeLaAG cells exposed or not
for 48 h to 40% AS fraction or to PDGF-BB were electrophoresed on
SDS-PAGE and analyzed by Western blot with CT-695 or 6E10 APP
antibodies or Gal4 antibody, as indicated. This demonstrated that the
exposure to 40% AS fraction or PDGF-BB results in a change of the size
of the APP-Gal4 bands, toward a major band of about 100 kDa, similar to
that of wild type Gal4 (indicated by an arrowhead), and
recognized by both Gal4 antibody and CT-695 antibody directed against
the C-terminal domain of APP. On the contrary, 6E10 antibody failed to
recognize the cleaved protein, thus demonstrating that it does not
contain the N-terminal
-amyloid epitope. The asterisk
indicates the APP-Gal4 bands. C, HeLaAG cells transfected
with G5BCAT vector and exposed to PDGF-BB or 40% AS fraction, as in
Fig. 3, were treated with 10 µM
-secretase inhibitor
compound X for 12 or 24 h, as indicated. Standard deviations of
triplicate experiments are reported.
-secretase activity, we treated HeLaAG cells exposed to PDGF or to
the 40% AS fraction with the
-secretase inhibitor compound X (25).
As shown in Fig. 4C, the treatment of HeLaAG cells exposed
to either PDGF or partially purified fraction with 10 µM
of the
-secretase inhibitor resulted in an almost complete abolishment of the effects on CAT accumulation. These results indicate
that PDGF, through the activation of its receptor, induces a
proteolytic cleavage of APP-Gal4, which requires the
-secretase activity.
View larger version (17K):
[in a new window]
Fig. 5.
Inhibition of Src TK activity prevents
APP-Gal4 cleavage in HeLaAG cells exposed to either PDGF-BB or
partially purified C6-conditioned medium. HeLaAG cells transiently
transfected with G5BCAT vector were exposed to 40 ng/ml recombinant
PDGF-BB or to 10 µg/ml of the 40% AS fraction, as reported in Fig.
4. These cells were also treated for 24 h before harvesting
with either 100 µM ERK inhibitor PD098059, 100 nM PI3K inhibitor wortmannin, 10 µM Src
inhibitor PP2, or with a PP2-like molecule, PP3, devoid of Src TK
inhibiting activity. Standard deviations of triplicate experiments are
reported.
View larger version (12K):
[in a new window]
Fig. 6.
The non-receptor Src TK and the small
G-protein Rac1 are responsible for the PDGF-induced cleavage of
APP-Gal4. A, HeLaAG cells transiently co-transfected
with G5BCAT vector and with vectors driving the expression of
constitutively active mutants of Src (SrcYF), Abl (Abl-PP), and Rac1
(RacQL) or with SrcYF plus a dominant negative mutant of Rac1 (RacN17).
When indicated, HeLaAG cells were transiently transfected with the
dominant negative mutants of Src (SrcYFKM) or of Rac (RacN17) and
treated for 24 h before harvesting with PDGF-BB or 40% AS
fraction. B, HeLaAG cells transiently co-transfected with
G5BCAT vector and with SrcYF or RacQL expression vectors and treated or
not with 10 µM -secretase inhibitor, compound X. Standard deviations of triplicate experiments are reported.
-secretase-dependent pathway,
HeLaAG cells were transfected with SrcYF or with RacQL, the
constitutively active mutants of these two proteins, and treated with
the
-secretase inhibitor compound X. As shown in Fig. 6B,
the
-secretase inhibitor almost completely abolished the effects of
SrcYF and RacQL transfections.
from Wild Type APP through an
Src-dependent Pathway--
The above reported results
indicate a clear dependence of the PDGF-Src-induced cleavage of APP
upon the
-secretase activity, but they don't allow us to
distinguish between
-secretase and BACE activities, whose actions
are known to precede the
-secretase-induced cleavage. To address
this point, we examined the effects of the PDGF-Src pathway on the
processing of APP, by measuring the accumulation of A
in cultured
cells, in which this pathway is activated or blocked. To do this,
HEK293 cells were transfected with APP695 alone or with
APP695 plus SrcYF. As shown in Fig.
7A, there is a significantly
increased accumulation of A
in the medium of cells expressing the
constitutively active form of Src. Furthermore, CHO cells stably
expressing APP695, which generate high levels of A
, were
treated with two concentrations of the inhibitor of Src TK, PP2. In
these conditions, A
generation is significantly decreased, whereas
the analogous molecule PP3, not affecting Src TK activity, was
completely ineffective (see Fig. 7B).
View larger version (10K):
[in a new window]
Fig. 7.
Src TK activity regulates
A generation from wild type
APP695. A, HEK293 cells were transiently
transfected with the vector encoding APP695 with or without
the vector driving the expression of the constitutively active SrcYF
mutant. 36 and 48 h after transfections, total A
peptide
present in the culture medium was measured by enzyme-linked
immunosorbent assay. In the case of the 36-h point of the cells
transfected with APP alone, some measurements were below the detection
limit (n.d., non-detectable). B, CHO cells stably
expressing APP695 were exposed to 5 or 20 µM
concentrations of the Src TK inhibitor PP2 or to 20 µM
PP3. The bars indicate the amount of total A
present in
the medium after 1, 3, and 12 h. The values are means of at least
triplicate experiments, and standard deviations are reported.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
peptide is an extensively studied phenomenon, due to its implication in the pathogenesis of Alzheimer's disease (AD). The great
effort to understand the machineries involved in the various types of
cleavages of APP resulted in the identification and in the molecular
characterization of two out three of the secretases, i.e.
- and
-secretases, and many preliminary results indicate that,
despite its complexity, also
-secretase is near to be understood. On
the contrary, the mechanisms regulating this proteolytic processing are
not completely understood. Here, we report experiments demonstrating that the
-
processing of APP is under a positive control by PDGF
through a pathway involving Src and Rac1.
generation (36, 37). On the
contrary, very little is known of the possible effects of the
activation of tyrosine kinase receptors on APP processing.
-receptor is expressed in neurons of various districts of mouse and
rat CNS. This expression, detected as early as postnatal day 1, is
observed during all the postnatal life, whereas the expression of PDGF
-receptor in oligodendrocytes is abundant during development, but is
restricted in the adult to few precursor cells (38). These results are
in agreement with several observations indicating a protective role for
PDGF in several neuronal cells (39-41). PDGF-A and
PDGF-B are constitutively expressed by neurons in
vivo (42), and this suggests further that these growth factors, which regulate proliferation and differentiation of oligodendrocytes (42), could also regulate the functions of the neurons themselves (38).
Src, and the related non-receptor TK Fyn, are expressed in the neurons,
are enriched in growth cones (43), and are involved in several neuronal
functions, such as for example Ig CAM-mediated neurite growth and
guidance (44). The three members of the Rho family of small GTPases,
Rho, Rac1, and Cdc42, are ubiquitously involved in actin cytoskeleton
regulation, affecting cell attachment and contraction, lamellipodia
formation, and filopodia formation, respectively (45). Their
involvement in the regulation of neuronal functions is well documented.
In particular, Rac has been implicated in neurite outgrowth and axonal
pathfinding (46). In addition to numerous in vitro
results, this is demonstrated by the expression of a constitutively
active form of Rac1 in Purkinje cells, which resulted in an ataxic
phenotype of mice that is accompanied by alterations of dendritic
spines (47). Accordingly, the phenotypes induced by combined mutations
of the three Rac GTPases of Drosophila are characterized by
defects of branching, guidance, and growth of axons (48).
-
processing is activated by Rac1 and
the well demonstrated function of Rac1 in the activation of p35/Cdk5
suggest a possible crucial role for this small G-protein in the
generation of the pathological signs of AD. In fact, the two histologic
hallmarks of the disease are A
accumulation in senile plaques and
the organization of hyperphosphorylated tau protein in fibrillary
tangles. It is well demonstrated that one of the two kinases involved
in anomalous tau phosphorylation is p35/Cdk5 (51), and therefore,
activation of Rac1 could, at the same time, increase A
generation
and cause tau hyperphosphorylation, leading to conditions that favor
plaque and tangle formation.
-secretase is not completely known, and little information is available on the regulation of BACE
activity; on this basis, it is hard to hypothesize mechanisms through
which these machineries could be activated. On the other hand, there
are experimental results suggesting that phosphorylation of APP does
not affect its processing. In fact, it was well documented that APP is
phosphorylated on Ser and Thr in vitro and in
vivo (59), but these post-translational modifications are not
involved in the regulation of APP cleavage (60). APP is also
phosphorylated at the level of Tyr-682 (APP695
isoform numbering) of its intracellular domain (8). One of the kinases
that is able to phosphorylate APP on Tyr-682 is the non-receptor
tyrosine kinase Abl. However, we showed here that the expression of a
constitutively active form of Abl does not affect APP-Gal4 processing
(see Fig. 6) and that, in cells expressing a mutant form of APP in
which Tyr-682 is substituted with a Phe residue, SrcYF induces an
increase of A
accumulation similar to that observed in cells
expressing wild type APP (data not shown).
-secretase could have a role in signal transduction (61). In
fact, we and others (10, 62) demonstrated that Fe65, one of the ligands
of APP cytodomain, is a nuclear protein and that APP functions as an
anchor that restricts Fe65 outside of the nucleus. Following APP
processing by
-secretase, the cytodomain of APP (AID) together with
Fe65 is translocated into the nucleus (10, 62-63). Fe65 and/or
AID·Fe65 complex, through the interaction with the
transcription factor LSF (9) or with the histone
acetyltransferase Tip60 (10), could regulate the transcription.
In support to this hypothesis, we found that Fe65 overexpression in the
nucleus regulates the transcription of the thymidylate synthase gene
driven by LSF (64). These findings suggest that PDGF, or other
molecules activating the Src-Rac1 cascade, could be signals that
trigger the cleavage of APP and, in turn, nuclear translocation of Fe65 and/or Fe65-AID, which regulate gene expression.
by neurons and that new possible targets for therapeutic interventions
in Alzheimer's disease could be found in this pathway. Furthermore,
the experimental system described in this report could be used to find
molecules that inhibit the PDGF-Src-Rac-induced processing of APP and
that, in turn, could be useful for the development of anti-AD drugs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Silvio Gutkind and
Mario Chiariello for the Src and Rac vectors, Daniela Barilà and
Giulio Superti-Furga for the Abl-PP vector, and Eddie Koo for the CHO
cells expressing APP. Véronique Hubert and Thierry Canton are
greatly acknowledged for A quantification.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the V Framework program (contract QLK6-1999-02238) EU, from the Italian Ministry of Health (Progetto Alzheimer), Miur-FIRB RBNE0IWY7B, from Biogem-Italy (to T. R.), and from MIUR-PRIN (to N. Z.).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.
§ Both authors contributed equally to this work.
¶ Recipient of a Biogem fellowship.
§§ To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, via S. Pansini 5, Napoli 80131, Italy. Tel.: 39-08-17-46-3131; Fax: 39-08-17-46-4359; E-mail: russot@dbbm.unina.it.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211899200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A, beta-amyloid
peptide;
AID, APP intracellular domain;
APP, amyloid precursor protein;
AS, ammonium sulfate;
BACE,
-site APP cleaving enzyme;
CAT, chloramphenicol acetyltransferase;
PTB, phosphotyrosine binding domain;
TK, tyrosine kinase;
PDGF, platelet-derived growth factor;
PDGF-R, PDGF
receptor;
CMV, cytomegalovirus;
DMEM, Dulbecco's modified Eagle's
medium;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline;
FPLC, fast-protein liquid chromatography;
MALDI-TOF, matrix-assisted
laser desorption ionization time-of-flight;
MAPK, mitogen-activated
protein kinase;
ERK, extracellular signal-regulated kinase;
PI3K, phosphatidylinositol 3-kinase;
AD, Alzheimer's disease;
PDGF-B, PDGF B subunit;
PDGF-BB, PDGF-B dimer;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine;
PP3, 4-amino-7-phenylpyrazol(3,4-d)pyrimidine;
sAPP, soluble
APP.
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