From the Centre for Neuronal Survival, Montreal Neurological Institute, and the Department of Neurology and Neurosurgery, McGill University, Montréal, Québec H3A 2B4, Canada
Received for publication, December 20, 2000, and in revised form, April 3, 2001
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ABSTRACT |
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The Akt kinase plays a crucial role in supporting
Trk-dependent cell survival, whereas the p75 neurotrophin
receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism
that p75NTR uses to promote cell death is not certain, but one
possibility is that p75NTR-dependent ceramide accumulation
inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test
this hypothesis, we developed a system for examining
p75NTR-dependent apoptosis and determined the effect of
p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than
decreased, Akt phosphorylation in a variety of cell types, including
human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase
activity. The p75NTR expression level required to elicit Akt
phosphorylation was much lower than that required to activate the JNK
pathway or to mediate apoptosis. We show that
p75NTR-dependent Akt phosphorylation was independent of
TrkA signaling, required active phosphatidylinositol 3-kinase, and was
associated with increased tyrosine phosphorylation of p85 and Shc and
with reduced cytosolic tyrosine phosphatase activity. Finally, we show
that p75NTR expression increased survival in cells exposed to
staurosporine or subjected to serum withdrawal. These findings indicate
that p75NTR facilitates cell survival through novel signaling cascades
that result in Akt activation.
The neurotrophins are a family of growth factors involved in the
survival, development, and death of specific populations of neurons and
non-neuronal cells. Nerve growth factor
(NGF),1 the prototypic
neurotrophin, is the best characterized member of this family, which in
mammals, also includes brain-derived neurotrophic factor,
neurotrophin-3, and neurotrophin-4/5 (1). The signal transduction
systems that mediate the diverse biological functions of the
neurotrophins are initiated by two categories of cell-surface
receptors: the Trk receptors and the p75 neurotrophin receptor
(p75NTR).
One of the main survival pathways for neuronal cell survival is
mediated by phosphatidylinositol 3-kinase (PI3K) and involves activation of the Akt serine/threonine kinase (2). Increased phosphatidylinositol 3,4,5-trisphosphate (PIP3)
production results primarily from relocalization of PI3K from the
cytosol to a juxtamembrane location that provides access to PIP
substrates. This redistribution of PI3K requires the association of the
SH2 domain within the p85 regulatory subunit of PI3K with
phosphorylated tyrosines present on activated cell-surface receptors or
on receptor-associated adaptor proteins (reviewed in Ref. 3).
Accumulation of PIP3 and its phospholipid phosphatase
product, phosphatidylinositol 3,4-bisphosphate, in the plasma membrane
creates docking sites for the pleckstrin homology domains of
phosphoinositide-dependent kinase-1 and Akt.
Phosphorylation of Akt on threonine 308 by phosphoinositide-dependent kinase-1 followed by autophosphorylation on serine 473 activates Akt
(4, 5) and allows the enzyme to facilitate survival by
phosphorylation of downstream substrates that may include Bad, Caspase-9, Forkhead family members, I p75NTR binds all neurotrophins with similar affinity and is a member of
the tumor necrosis factor receptor (TNFR) superfamily (13, 14). Current
data suggest that the main physiological functions of p75NTR are to
regulate Trk receptor activation and signaling (15-19) and to activate
Trk-independent signal transduction cascades involving sphingomyelinase
(20-22), nuclear factor- Activation of cell death cascades can result from suppression of
signaling pathways that normally support survival. In some systems,
sphingomyelinase activation results in a ceramide-dependent decrease in the generation of PIP3 and a subsequent
reduction in Akt activity (42, 43); and in others, ceramide reduces Akt
activity through specific dephosphorylation of serine 473 (44). Since
p75NTR activates sphingomyelinase in a
neurotrophin-dependent manner, we have determined if p75NTR
activation can suppress Akt and thereby facilitate apoptosis. Our
results show that p75NTR does indeed regulate Akt; but contrary to our
expectations, we found that p75NTR increases Akt activation through a
Trk-independent pathway that requires PI3K and show that p75NTR
expression suppresses apoptosis. Although high levels of p75NTR will
mediate cell death, the p75NTR expression level required to elicit Akt
phosphorylation is much lower than that required to activate the JNK
pathway or to mediate apoptosis. The effect of p75NTR on Akt correlates
with increased tyrosine phosphorylation of the p85 regulatory subunit of PI3K and of Shc adaptor proteins, suggesting that PTPase inhibition may play a role in this effect. Consistent with this, p75NTR expression results in reduced cytosolic tyrosine phosphatase activity. These data
indicate that a physiological role of p75NTR is to enhance cell
survival through an Akt-dependent pathway.
Materials--
NGF was purchased from Collaborative Research
(Bedford, MA); cell culture reagents were from BioWhittaker, Inc.
(Walkersville, MD); and all other reagents were from Sigma, ICN
Biochemicals (Costa Mesa, CA), or Calbiochem unless otherwise indicated.
Preparation of Recombinant Adenoviruses--
pAd-CMV5-F1
containing full-length rat p75NTR (45), the p75NTR intracellular domain
(46), or the p75NTR intracellular domain modified to contain an
N-terminal myristoylation tag derived from Hck, an Src-related kinase
(47), was cotransfected with replication-defective adenoviral DNA
(Quantum Biotechnologies, Laval, Québec, Canada) into 293A cells.
Crude viruses derived from viral plaques were used to infect 293A
cells. p75NTR expression was confirmed by immunoblot analysis, and
positive plaques were repurified twice by limiting dilution.
Recombinant adenoviruses were amplified in 293A cells, purified on
sucrose gradients, and titered by plaque assay in 293A cells. Control
recombinant adenoviruses expressing Cell Culture--
The rat pheochromocytoma cell lines PC12 and
PC12nnr5 were maintained in 7.5% CO2 at 37 °C in
Dulbecco's modified Eagle's medium with 5% bovine calf serum (BCS),
5% horse serum, 2 mM L-glutamine, and 100 µg/ml penicillin/streptomycin. Normal and Niemann-Pick human
fibroblasts (obtained from the NIGMS/Human Genetic Mutant Cell
Repository, Camden, NJ), MG87-3T3, HeLa, COS-7, and A875 cells were
maintained in Dulbecco's modified Eagle's medium containing 10% BCS.
Doxycycline-inducible p75NTR-expressing MG87-3T3 fibroblasts (TIMp75-3)
were produced and maintained as described previously (24).
Immunoblotting--
Immunoblotting for total and phosphorylated
proteins was performed using rabbit polyclonal antibodies from New
England Biolabs, Inc. (Beverly, MA) or from Upstate Biotechnology, Inc.
(Lake Placid, NY). p75NTR immunoreactivity was detected using
p75NTR-B1, a rabbit polyclonal antibody directed against a glutathione
S-transferase fusion protein containing amino acids 276-425
of the intracellular domain of rat p75NTR (Babco, Berkeley, CA)
(46, 48). Protein content from cell lysates was normalized using the
BCA assay (Pierce), and 10-25 µg of protein was solubilized in
Laemmli sample buffer (49), separated by SDS-polyacrylamide gel
electrophoresis, and electroblotted onto nitrocellulose. Blocking and
secondary antibody incubations of immunoblots were performed in
Tris-buffered saline/Tween (10 mM Tris (pH 7.4), 150 mM NaCl, and 0.2% Tween 20) supplemented with 5% (w/v)
dried skim milk powder. Primary antibody incubations were performed in
Tris-buffered saline/Tween supplemented with 5% bovine serum albumin.
For 4G10, 2% (w/v) bovine serum albumin was used for the blocking
step. Horseradish peroxidase-conjugated donkey anti-rabbit IgG,
horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA), or horseradish
peroxidase-conjugated protein A was used at a dilution of 1:5000.
Immunoreactive bands were detected using enhanced chemiluminescence
(PerkinElmer Life Sciences) according to the manufacturer's instructions.
Transfection and Subcellular Fractionation--
COS-7
cells were transfected with a control plasmid or with plasmid encoding
the p75NTR intracellular domain or the p75NTR intracellular domain
modified to contain a myristoylation tag. Forty-eight hours after
transfection, cells were scraped from plates in cold phosphate-buffered
saline (PBS), centrifuged for 5 min at 2000 × g, and
then resuspended in 15 ml of HES buffer (20 mM HEPES (pH
7.4), 1 mM EDTA, 255 mM sucrose, 10 µg/ml
leupeptin, 25 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). Cells were homogenized in a
glass-on-Teflon homogenizer with 10 strokes at 1200 rpm and then
triturated twice using a 25-gauge needle. An aliquot was set aside as
the initial lysate. The lysate was centrifuged at 19,000 × g for 20 min; the resulting pellet was designated membrane,
and the supernatant was designated cytosol. All fractions were
resuspended in HES buffer containing 1% Nonidet P-40 and analyzed for
protein concentration, and equivalent amounts were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide
Survival Assays--
Analysis of cell survival was performed using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which was
added at a final concentration of 1 mg/ml for 4 h following a 48-h
infection. The reaction was ended by the addition of 1 volume of
solubilization buffer (20% SDS, 10% dimethylformamide, and 20%
acetic acid). After overnight solubilization, specific and nonspecific
absorbances were read at 570 and 630 nm, respectively. Each condition
was tested six times, and results were analyzed for statistical
significance by multiple analysis of variance.
Immunoprecipitation--
Twenty-four hours after infection,
cells were washed in cold Tris-buffered saline and lysed in Nonidet
P-40 lysis buffer (10 mM Tris (pH 8.0), 150 mM
NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate).
Immunoprecipitation was performed at 4 °C using polyclonal
anti-pan-Trk 203 antibody (gift of David Kaplan, Montreal
Neurological Institute, Montreal, Canada), polyclonal anti-Shc antibody
(gift of Jane McGlade, University of Toronto, Toronto, Ontario,
Canada), or polyclonal anti-p85 antibody (Upstate Biotechnology, Inc.).
Complexes were precipitated using 45 µl of protein A-Sepharose
(Amersham Pharmacia Biotech), which was added for 90 min at 4 °C and
then subjected to multiple washes. For wheat germ agglutinin (Amersham
Pharmacia Biotech) precipitation, beads were added to Nonidet
P-40-extracted protein samples for 2 h at 4 °C, followed by
centrifugation and multiple washes. Samples were lysed in Laemmli
sample buffer and analyzed by immunoblotting as described above.
Apoptotic Assays--
Apoptotic cell death was quantified using
annexin V binding and FACS analyses. Briefly, cells were harvested
using PBS with 2 mM EDTA and washed twice in PBS
supplemented with 2% BCS. After the last wash, the cells were
resuspended in 0.1 ml of PBS containing 1 µg/ml fluorescein
isothiocyanate-conjugated annexin V (Becton Dickinson, Mountain
View, CA) and incubated for 15 min in the dark at room temperature. PBS
(0.3 ml) was added to each tube, and cells were analyzed on a FACScan
flow cytometer (Becton Dickinson-Pharmingen). For FACS analysis of
cells with sub-G1 DNA content, cells were harvested and
resuspended in 50% ethanol and PBS and left on ice for 15 min. An
underlayer of 1 volume of cold BCS was added, and cells were spun at
250 × g for 5 min. Cells in the resulting pellet were
resuspended in blocking buffer consisting of PBS containing 2% bovine
serum albumin and 2% BCS and then incubated for 30 min on ice.
Antibody p75NTR-B1 was added at a dilution of 1:500, and the incubation
was continued for an additional 30 min. Cells were washed three times
in blocking buffer and then incubated for 30 min in blocking buffer
supplemented with a 1:500 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.). Cells were washed three times and resuspended in
0.1 ml of PBS with 0.1 mg/ml RNase A for 15 min at room temperature. PBS (0.3 ml) with 25 µg/ml propidium iodide was added to the cells, incubated for 15 min, and then analyzed on a FACScan. Each condition was tested in triplicate, and results were analyzed for statistical significance by multiple analysis of variance.
Protein-tyrosine Phosphatase Assays--
Twenty-four hours after
infection, PC12nnr5 cells were washed twice in cold HEPES-buffered
saline to remove free phosphate and then harvested in 1 ml of
suspension buffer (50 mM HEPES, 150 mM NaCl, 2 mM EDTA, 0.25 M sucrose, 1 mM
dithiothreitol, 10 µg/ml leupeptin, 25 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), sonicated, and spun at
1000 × g for 5 min to remove intact mitochondria and
nuclei. Cytosolic and membrane fractions were separated by spinning at
100,000 × g for 60 min at 4 °C. The membrane pellet
was resuspended for 30 min in 1 ml of suspension buffer supplemented
with 1% Triton X-100 and then spun at 1000 × g for 5 min to remove insoluble contaminants. Endogenous phosphate was removed
from membrane and cytosolic fractions by buffer exchange in
Centricon-10 columns (Amicon, Inc., Beverly, MA). Protein assays were
performed on the resulting fractions, and the tyrosine-phosphorylated substrate END(pY)INASL was added to 25 µg of protein to a final concentration of 100 µM. The reaction was stopped after
30 min at room temperature by the addition of 1 volume of stopping
solution (0.02% malachite green, 0.5% ammonium molybdate
tetrahydrate, and 0.1% polyvinyl alcohol). Absorbance at 630 nm was
read after a 60-min incubation. As a control, sodium orthovanadate was
added to a parallel set of samples to ensure the presence of specific tyrosine phosphatase activity. Background absorbance for parallel samples that did not receive the substrate was subtracted as
contaminating free phosphate. Each condition was tested in triplicate,
and results were analyzed for statistical significance by multiple
analysis of variance.
Recombinant Adenoviruses Encoding p75NTR Activate JNK and Induce
Cell Death of PC12nnr5 Cells--
The actions of p75NTR are complex
and not well understood. This is due in part to the lack of cellular
systems in which p75NTR-dependent signaling events can be
reliably observed. Previous work from our group (46) and others (50)
has shown that overexpression of the intracellular domain of TNFR
superfamily members will constitutively activate downstream signaling
pathways. To reliably activate p75NTR signaling cascades in a variety
of cellular circumstances, recombinant adenoviruses encoding
full-length p75NTR, the intracellular domain of p75NTR (p75ICD), or a
myristoylated form of the intracellular domain of p75NTR that is
targeted to the plasma membrane (p75mICD) (Fig.
1B) were produced. Recombinant
adenoviruses encoding LacZ or green fluorescent protein were used as
controls for all studies. To validate signaling properties of these
p75NTR recombinant viruses, we initially tested their effects on the
survival of PC12nnr5 cells (which express endogenous p75NTR, but not
TrkA). Fig. 1A shows that LacZ virus had no effect on cell
survival, whereas viruses encoding p75NTR or p75ICD were cytotoxic at a
multiplicity of infection (m.o.i.) of 200. Overexpression of p75mICD
induced cytotoxicity at lower multiplicities of infection than that of p75NTR or p75ICD, suggesting that membrane localization of the intracellular domain may be important for p75NTR-dependent
apoptosis. p75NTR-dependent cell death was not an artifact of
protein overexpression or viral infection since equivalent quantities
of LacZ adenovirus had no effect on cell survival. Several reports have
shown that p75NTR activation can lead to JNK activation (26-28); and
consistent with this, we found that expression of p75mICD (Fig.
1C), p75ICD, and p75NTR increased phosphorylation of JNK on
threonine 183 and tyrosine 185 and increased c-Jun phosphorylation on
serine 73. Equivalent levels of LacZ virus did not alter JNK or c-Jun
phosphorylation (data not shown). The level of c-Jun protein was also
elevated at high p75NTR expression levels, likely due to autoregulation of c-jun transcription (51) during the relatively long
infection period (48 h). To demonstrate the specificity of the JNK
activation initiated by p75NTR, we analyzed phosphorylation of the
transcription factor CREB, which lies downstream of protein kinase A,
and assessed the phosphorylation status of MKK3/6, which activates p38
MAPK. Neither CREB (Fig. 1C) nor MKK3/6 (data not shown)
phosphorylation was altered by p75NTR expression . These results show
that p75NTR signaling results in specific activation of the JNK pathway
and promotes cell death and therefore demonstrate that the p75NTR recombinant adenoviruses show the expected constitutive signaling properties.
p75NTR Activates Akt--
Recent reports have shown that ceramide
inhibits PI3K activity, reduces cellular PIP3 levels, and
thereby inhibits Akt activity (42, 43). p75NTR activates
sphingomyelinase upon neurotrophin binding (20, 21), and we
hypothesized that p75NTR could facilitate apoptosis by attenuating
PIP3 production and reducing Akt activity. To determine if
p75NTR alters Akt activity, PC12nnr5 cells were infected with
recombinant virus expressing each of the three p75NTR isoforms or LacZ
and analyzed for Akt activation using phospho-specific antibodies
directed against Akt serine 473 (Ser473), an Akt
autophosphorylation site that correlates with Akt kinase activity (5).
Surprisingly, expression of full-length p75NTR, p75ICD (Fig.
2), or p75mICD (see Figs. 3B
and 6B) resulted in significant increases in the
phosphorylation of Akt on Ser473, whereas control
adenovirus expressing LacZ had no effect on Akt Ser473
phosphorylation. As noted above, relatively high levels of p75NTR expression were required to observe JNK activation, c-Jun
phosphorylation, and apoptosis, but much lower levels of p75NTR
expression were sufficient to induce Akt phosphorylation.
p75NTR-mediated reduction of Akt phosphorylation was never observed,
even in cells exposed to high titers of p75NTR recombinant adenovirus.
The effects of ligand binding to p75NTR on Akt activation in the
presence and absence of virus were also tested; treatment of control
and infected cells with NGF, brain-derived neurotrophic factor, or
neurotrophin-3 at various concentrations and time courses had no effect
on the p75NTR adenovirus-induced phosphorylation of Akt in PC12nnr5
cells (data not shown). Therefore, these data indicate that p75NTR
expression activates Akt in a ligand-independent manner.
p75NTR-induced Akt Phosphorylation Is Trk-independent and Does Not
Require Acidic Sphingomyelinase--
The TrkA receptor is a potent
activator of Akt, and our previous results (15) and those of others
(16, 52) have shown that p75NTR can increase the response of TrkA to
limiting NGF concentrations. It was therefore possible that the
p75NTR-dependent Akt phosphorylation observed is secondary
to activation of low levels of TrkA that may be present in PC12nnr5
cells. To address this, PC12nnr5 cells were first examined to determine
if they express NGF-responsive TrkA. Fig.
3A shows that activated TrkA was not detected in PC12nnr5 cells under conditions in which
TrkA was readily detected in normal PC12 cells. We then tested
K252a, a specific TrkA inhibitor, for its ability to block
p75NTR-mediated Akt phosphorylation using recombinant viruses encoding
p75ICD and p75mICD, which are incapable of binding ligand. LacZ,
p75ICD, and p75mICD adenoviruses were used at m.o.i. = 25 in these
experiments since this infection level activated Akt (Fig. 2), but did
not induce cell death (Fig. 1). Fig. 3B shows that K252a
completely blocked NGF-mediated Akt phosphorylation in PC12 cells, but
had no effect on p75ICD- and p75mICD-induced Akt phosphorylation in either PC12nnr5 or PC12 cells. Therefore, p75NTR-mediated activation of
Akt occurs independently of TrkA signaling.
PC12nnr5 cells express endogenous p75NTR, and it is conceivable that
p75NTR overexpression may mediate an increase in Akt phosphorylation by
disrupting signaling from endogenous p75NTR. To address this, the
effects of p75NTR on Akt phosphorylation were determined in a variety
of cell types that do not express p75NTR or TrkA. p75NTR expression in
MG87-3T3 fibroblasts (m.o.i. = 100) (Fig. 3C) or HeLa cells
(data not shown) resulted in increased Akt phosphorylation, similar to
that observed in PC12nnr5 cells. These results indicate that the effect
of p75NTR on Akt phosphorylation does not involve disruption of
endogenous p75NTR signaling.
Recombinant adenoviruses have significant effects on cellular
physiology, and we therefore sought additional means to confirm that
p75NTR expression increases Akt activation. TIMp75-3 is an MG87-derived
cell line in which expression of p75NTR is tightly regulated through
the addition of doxycycline (24) and therefore could be used to confirm
that p75NTR overexpression results in Akt phosphorylation. Cells
incubated with 2.5 µg/ml doxycycline for 24 h showed the
expected increase in p75NTR expression, and this correlated with a rise
in Akt Ser473 phosphorylation (Fig. 3D).
Therefore, two independent means of p75NTR expression result in Akt
activation in different cell types. The relatively modest increase in
Akt phosphorylation observed likely reflects the fact that only a
subpopulation of TIMp75-3 cells show robust doxycycline-induced p75NTR
expression (see Fig. 5B).
Neurotrophin binding to p75NTR increases sphingomyelinase activity, and
one possible explanation for the effect of p75NTR on Akt is that
unliganded p75NTR functionally inactivates cellular sphingomyelinase,
thereby reducing cellular ceramide levels and causing a consequent
increase in PI3K activity, PIP3 levels, and Akt
phosphorylation. To address this, we compared the effects of p75NTR
overexpression on Akt activation in normal and Niemann-Pick fibroblasts. Niemann-Pick fibroblasts are deficient in acidic sphingomyelinase, which is the only form of sphingomyelinase activated by p75NTR in PC12 cells.2
Fig. 4 shows that p75NTR and p75ICD
induced Akt phosphorylation in primary human fibroblasts derived from
control and Niemann-Pick patients, suggesting that inactivation of
acidic sphingomyelinase activation is not involved in this p75NTR
response.
Expression of p75NTR Increases Cell Survival--
These data are
consistent with the hypothesis that p75NTR produces biphasic autonomous
responses. High levels of p75NTR signaling result in JNK activation,
c-Jun phosphorylation, and cell death, and lower levels of p75NTR
signaling induce alternative pathways that include Akt activation and
survival. To examine this, PC12nnr5 cells were infected with LacZ,
p75NTR, or p75ICD adenovirus (all at m.o.i. = 25) and exposed to 0.5 µM staurosporine for 18 h, and levels of cellular
apoptosis were determined by assessing annexin V binding by FACS
analysis. Fig. 5A shows that
infection of PC12nnr5 cells with p75NTR and p75ICD viruses
significantly reduced the incidence of apoptosis compared with
Me2SO-treated cells or cells infected with control LacZ
virus.
To extend these results to other cellular models, TIMp75-3 cells
were analyzed for p75NTR-mediated survival properties. TIMp75-3 cells
were produced from the MG87 cell line, which undergoes rapid cell death
in the absence of serum-derived growth factors. TIMp75-3 cells were
treated with 2.5 µg of doxycycline for 36 h (to induce p75NTR
expression) and then deprived of serum for 18 h. FACS analysis was
performed to assess p75NTR expression and apoptosis. Fig. 5B
clearly shows that the TIMp75-3 population that expressed p75NTR was
much less susceptible to cell death induced by serum deprivation, indicating that p75NTR promotes survival under these circumstances.
Activation of Akt by p75NTR Requires Active PI3K--
To begin to
determine the mechanisms used by p75NTR to activate Akt, the effect of
PI3K inhibitors on p75NTR-induced Akt phosphorylation was examined.
LY294002 and wortmannin were first tested for their ability to block
Akt phosphorylation induced by NGF in PC12 cells. Fig.
6A shows that, as expected,
both inhibitors efficiently reduced NGF-induced Akt activation. Both
inhibitors also completely blocked Akt phosphorylation induced by the
p75mICD (Fig. 6B) or p75ICD or p75NTR (data not shown)
recombinant adenovirus in PC12nnr5 cells and in A875 human melanoma
cells. Therefore, PI3K activity is required for p75NTR-mediated
activation of Akt.
p75NTR Expression Increases Tyrosine Phosphorylation of p85 and
Shc--
Activated receptor tyrosine kinases increase PIP3
production largely by allowing PI3K proximity to the plasma membrane
through SH2 domain-mediated interactions of the p85 regulatory subunit with receptor or with receptor-associated adaptor proteins such as Shc.
To determine if Shc could contribute to the p75NTR-mediated increase in
Akt phosphorylation, the tyrosine phosphorylation level of
immunoprecipitated Shc was analyzed in cells expressing p75ICD or
control adenovirus. Fig. 7A
shows that phosphorylation of all three Shc isoforms (p66, p52, and
p46) was increased relative to controls and that this effect was
potentiated when tyrosine phosphatase activity (PTPase) was inhibited
by treating the cells with sodium orthovanadate for 1 h prior to
harvesting. A 160-kDa protein that co-immunoprecipitated with Shc also
showed increased tyrosine phosphorylation in response to p75NTR
overexpression; this protein is likely SHIP, a 160-kDa lipid
phosphatase that binds activated Shc with high affinity (53).
Experiments were also performed to determine if the p85 subunit of PI3K
shows a similar p75NTR-dependent increase in tyrosine
phosphorylation. Fig. 7B shows that full-length p75NTR, but
not green fluorescent protein, strongly increased tyrosine
phosphorylation of p85. These results indicate that p75NTR activates a
PI3K/Akt pathway by increasing tyrosine phosphorylation of adaptor
proteins that relocalize PI3K to the plasma membrane. To determine if
the phosphotyrosine content of any cell-surface proteins is altered by
p75NTR expression, PC12nnr5 cells were infected with p75ICD recombinant
adenovirus, and cell-surface glycoproteins were precipitated using
Sepharose-conjugated wheat germ agglutinin and assayed for
phosphotyrosine content by 4G10 immunoblotting. Fig. 7C
shows that p75NTR expression specifically increased tyrosine
phosphorylation of a 120-kDa protein, particularly in cells pretreated
with sodium orthovanadate.
p75NTR Decreases Protein-tyrosine Phosphatase Activity--
Our
results show that p75NTR expression increased tyrosine phosphorylation
of several proteins, particularly in the presence of orthovanadate, a
nonspecific competitive PTPase antagonist. p75NTR has no intrinsic
enzymatic activity, and its effect on phosphorylation must be due to
regulatory interactions with proteins capable of increasing kinase
activity, decreasing PTPase activity, or some combination of the two. A
physical interaction between p75NTR and the FAP PTPase has recently
been reported (38), raising the possibility that cellular tyrosine
phosphatase activity could be modulated by p75NTR. To test this,
PC12nnr5 cells were infected with full-length p75NTR, p75ICD, or LacZ
adenovirus at m.o.i. = 25, subjected to subcellular fractionation, and
analyzed for cytosolic and membrane-bound PTPase activities. Fig.
8 shows that expression of p75NTR or
p75ICD resulted in a significant decrease in cytosolic PTPase activity,
whereas membrane-associated PTPase activity was unchanged by p75NTR
expression. These results indicate that p75NTR is capable of inhibiting
cytosolic PTPase(s) and suggest that increases in the phosphotyrosine
content mediated by p75NTR may be secondary to alterations in PTPase
activity.
We have demonstrated that p75NTR expression leads to
Trk-independent, PI3K-dependent Akt phosphorylation. High
levels of p75NTR expression increase JNK and c-Jun phosphorylation and
promote apoptosis, yet lower p75NTR expression levels are associated
with activation of Akt and suppression of apoptosis induced by distinct stressors. p75NTR expression levels that potentiate Akt phosphorylation and survival increase the phosphotyrosine content of several cellular proteins, including p85 and Shc, suggesting that p75NTR affects the
activity of tyrosine kinases or PTPases. Consistent with this, we
demonstrate that p75NTR expression is associated with a decrease in
cytosolic PTPase activity.
Many studies have demonstrated that p75NTR can facilitate apoptosis.
Our earlier work (46) has shown that overexpression of the p75NTR
intracellular domain within neurons of transgenic mice results in
dramatic loss of peripheral and central neurons, and Barde and
co-workers (30, 55) has shown that embryonic retinal cells that
express p75NTR undergo cell death that can be prevented by the
application of antibodies against either NGF or the p75NTR
extracellular domain. Genetically altered mice rendered null at p75NTR
or NGF loci show deficits in developmental apoptosis within the retina
and spinal cord (31), and p75NTR can facilitate apoptosis of cultured
rat oligodendrocytes (29) and sympathetic neurons (27). The precise
pathways that p75NTR activates to induce apoptosis are unclear, but
JNK, caspase activation, and increased p53 levels have been observed in
some systems (26-28, 32). To reliably activate p75NTR signaling
cascades, we created recombinant adenoviruses that encode either
full-length p75NTR or the p75NTR intracellular domain. As expected from
our earlier work in transgenic mice (46), adenovirus-mediated
overexpression of p75ICD resulted in cellular apoptosis that was
associated with increased JNK activity and c-Jun phosphorylation.
Expression of p75NTR or myristoylated p75ICD gave similar results, with
p75mICD proving a particularly potent apoptotic inducer. These reagents will be useful for studies designed to identify specific signaling events in the p75NTR apoptotic cascade.
Neurotrophin binding to p75NTR results in the activation of
sphingomyelinase and the production of ceramide (20-22, 29, 56). Ceramide generated by p75NTR activation may inhibit Trk receptor activation (57), activate JNK (29, 58, 59), and affect neuronal
differentiation (22). In some systems, sphingomyelinase activation
results in a ceramide-dependent decrease in
PIP3 production and a subsequent reduction in Akt activity
(42, 43), and our initial hypothesis was that
p75NTR-dependent ceramide accumulation would suppress PI3K
activity and thereby reduce Akt activation. However, overexpression of
p75NTR resulted in ligand-independent activation of Akt in multiple
cell types. Indeed, Akt was activated even at p75NTR expression levels
that facilitate apoptosis, indicating that when the apoptotic pathway
is activated, it can override the pro-survival effect of Akt.
The activation of Akt by p75NTR requires active PI3K and correlates
with increases in the phosphotyrosine content of several proteins,
including the adaptor protein Shc, the p85 regulatory subunit of PI3K,
and a 120-kDa cell-surface protein. The increased phosphotyrosine
content of these proteins correlates with reduced cytosolic PTPase
activity in the presence of p75NTR. p75NTR-mediated inhibition of a
cytosolic PTPase may therefore be a proximal event in the signaling
cascade that may allow a Shc·PI3K complex to associate with the
plasmalemma and increase PIP3 production. Additional studies will be required to identify specific phosphatase(s) inhibited by p75NTR, but one candidate is FAP, which physically interacts with
p75NTR when overexpressed in 293T cells (38). FAP also binds human Fas
(60), and Fas-mediated apoptosis can be suppressed by FAP (61, 62)
through an unknown mechanism. An alternative mechanism that may account
for p75NTR-mediated Akt activation involves a link between TRAF
proteins and Src kinases. The TRANCE receptor is a member of the TNFR
superfamily that activates survival pathways in osteoclasts in part by
activating Akt, and a recent study has found that a complex of Src
kinase and TRAF-6 is required for this effect (63). p75NTR interacts
with several members of the TRAF family, including TRAF-6 (25, 34),
raising the possibility that this signaling path may also contribute to
p75NTR-mediated Akt activation. Future experiments specifically
examining FAP and Src signaling will be required to reveal the relative
contributions of each of these pathways to p75NTR-mediated Akt activation.
The signaling mechanisms employed by p75NTR are not well
understood, and the relationship of neurotrophin binding to p75NTR action remains unclear. All neurotrophins activate sphingomyelinase when bound to p75NTR (20, 21), but only NGF is capable of inducing
apoptosis and nuclear factor- Cytotoxic effects are observed when p75NTR is highly
overexpressed, but much lower expression levels of full-length p75NTR and the intracellular domain mutants elicit Akt phosphorylation and
enhance survival. The canonical view of p75NTR action is that receptor
signaling is activated by ligand binding, but recent studies on other
TNFR superfamily members suggest an alternative paradigm for p75NTR
signaling. Lenardo and co-workers (66, 67) have recently shown that
some TNFR superfamily members must pre-assemble into cell-surface
oligomers before binding ligand. Many investigators have observed
oligomeric p75NTR in the absence of ligand in a variety of preparations
(for an early example, see Ref. 54), consistent with the possibility
that p75NTR may also pre-assemble into oligomers. With Fas and TNFR-1,
ligand binding produces a conformational shift that enables specific
signaling events, but does not alter the oligomeric nature of the
receptor complex. Thus, the role of ligand is to shift the
pre-assembled receptor complex to a different signaling mode. All three
p75NTR constructs employed in our studies elicited Akt activation when
produced at low expression levels, but much higher levels of expression were required for apoptotic signaling. We favor the hypothesis that
these signaling events reflect distinct p75NTR signaling complexes and
that oligomeric p75NTR exists in at least two distinct signaling
complexes; in the ligand-free state, p75NTR will constitutively activate survival pathways that involve Akt, and when bound by ligand,
it activates signaling pathways that result in cellular apoptosis.
In conclusion, we have identified a novel p75NTR signaling pathway that
results in phosphorylation of Akt and that enhances cell survival.
These data suggest that the autonomous signaling role of p75NTR may be
broader than previously considered, with p75NTR capable of signaling
pathways that support survival and death under different cellular circumstances.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinase, and glycogen synthase kinase-3 (6-12).
B (23-25), and JNK (26-28). Several
findings indicate that NGF binding to p75NTR can initiate a cell death
cascade in some cell types. For example, NGF treatment of embryonic
retinal cells or postnatal oligodendrocytes that express p75NTR, but
not TrkA, increases cellular apoptosis (29-31). The precise signaling
pathway(s) used by p75NTR to activate cell autonomous death cascades
remain unclear, but may involve activation of caspase-1, -2, and -3 (32) as well as cyclin-dependent kinases (33). A number of
cytosolic proteins that interact directly with the p75NTR intracellular domain have been identified, including TRAF proteins (25, 34), caveolin (35), SC-1 (36), NRIF (37), FAP-1 (38), NADE (39), RhoA
(40), and NRAGE (41), but linking each of these to precise p75NTR
signaling cascades remains a major challenge.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (LacZ) or green
fluorescent protein were generated using the same viral backbone and
purification techniques as for the p75NTR viruses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Overexpression of p75NTR in PC12nnr5 cells
induces cell death. A, PC12nnr5 cells were infected
with recombinant adenoviruses expressing LacZ, full-length p75NTR, the
intracellular domain of p75NTR (p75ICD), or the myristoylated p75NTR
intracellular domain (p75mICD) at increasing multiplicities of
infection and, after 48 h, were assayed for survival using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay as described under "Experimental
Procedures." B, p75mICD is enriched in cell membranes, but
p75ICD is cytosolic. COS-7 cells were transiently transfected with
plasmids encoding either p75ICD (ICD) or p75mICD
(mICD) and, after 48 h, fractionated as described under
"Experimental Procedures." C indicates control.
C, PC12nnr5 cells that were infected and incubated under the
same conditions as described for A were lysed and analyzed
for c-Jun (phospho-Ser73 (pS73)), JNK
(phospho-Thr183/Tyr185), and CREB
(phospho-Ser133) phosphorylation by immunoblotting. Results
shown in A are represented as the means ± S.E. of
three independent experiments, and the experiment in C was
repeated twice with similar results.
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[in a new window]
Fig. 2.
Expression of p75NTR in PC12nnr5 cells
increases Akt Ser473 phosphorylation. PC12nnr5 cells
were infected with recombinant adenovirus expressing LacZ, full-length
p75NTR, or the intracellular domain of p75NTR (p75ICD) at different
multiplicities of infection. Cells were harvested 24 h after
infection and analyzed for Akt levels, Akt serine 473 phosphorylation,
LacZ expression, and p75NTR expression by immunoblotting. Experiments
were repeated three times with similar results. pS473,
phospho-Ser473; -Gal,
-galactosidase.
View larger version (35K):
[in a new window]
Fig. 3.
p75NTR-induced Akt phosphorylation is
Trk-independent. A, PC12 or PC12nnr5 cells were treated
for 15 min with 10 ng/ml NGF and lysed in Nonidet P-40 lysis
buffer as described under "Experimental Procedures." Trk receptors
were immunoprecipitated (IP) using the anti-pan-Trk 203 antibody. Protein lysates were analyzed by immunoblotting for levels of
total and phosphorylated Akt, phosphotyrosine (pTyr)
content, and TrkA levels within immunocomplexes. B, PC12 or
PC12nnr5 cells were infected with LacZ, p75ICD (ICD), or
p75mICD (mICD) adenovirus at m.o.i. = 25 for 24 h and
then treated with the Trk inhibitor K252a (200 nM) for 30 min, followed by 50 ng/ml NGF for 15 min before harvest. Comparison
between the two cell lines shows that the Trk inhibitor K252a
effectively inhibited Akt phosphorylation induced by NGF in PC12 cells,
but had no effect on p75NTR-induced activation of Akt in PC12 or
PC12nnr5 cells. C, MG87-3T3 cells were infected with
recombinant adenovirus encoding p75NTR or p75ICD at m.o.i. = 100, harvested 24 h later, and analyzed for Akt levels and Akt
Ser473 (pS473) phosphorylation. D,
TIMp75-3 cells were treated with 2.5 µg/ml doxycycline
(DOX) for 24 h; harvested; and analyzed for Akt levels,
Akt Ser473 phosphorylation, and p75NTR expression.
Experiments shown in A and B were repeated twice,
and those in C and D were repeated three times,
all with similar results.
View larger version (59K):
[in a new window]
Fig. 4.
p75NTR-mediated Akt phosphorylation does not
require acidic sphingomyelinase activity. Overexpression of p75NTR
or p75ICD in control human (upper panel) or Niemann-Pick
(lower panel) fibroblasts induced Akt phosphorylation. High
viral multiplicities of infection (m.o.i. = 100-200) were due to the
low infectability of these lines (data not shown). Results shown were
repeated twice with similar results. pS473,
phospho-Ser473.
View larger version (19K):
[in a new window]
Fig. 5.
Expression of p75NTR facilitates cell
survival. A, PC12nnr5 cells were infected with
recombinant adenovirus encoding LacZ, p75ICD, or p75NTR at m.o.i. = 25;
incubated for 24 h; and then treated with 0.5 µM
staurosporine or vehicle (dimethyl sulfoxide (DMSO)) for
18 h and analyzed for annexin V binding. A minimum of 20,000 cells
were analyzed for each condition shown. B, TIMp75-3 cells
were treated with 2.5 µg/ml doxycycline (DOX) for 36 h and serum-deprived for 18 h. Flow cytometry was carried out to
determine p75NTR expression levels (using antibody p75NTR-B1) and to
determine the proportion of cells with sub-G1 DNA content
using propidium iodide staining. A minimum of 30,000 cells were
analyzed by flow cytometry for each condition. Statistically
significant differences determined by multiple analysis of variance are
indicated with asterisks in A and B
(*, p < 0.05; **, p < 0.001). Data in
A and B represent the means ± S.E. of three
independent experiments.
View larger version (49K):
[in a new window]
Fig. 6.
PI3K activity is required for p75NTR-induced
phosphorylation of Akt. A, PC12 cells were treated with
50 ng/ml NGF and 100 nM wortmannin (W), 20 µM LY294002 (LY), or equivalent quantity of
DMSO (D) for 15 min prior to lysis and analyzed by
immunoblotting for Akt levels and for Akt Ser473 (pS473)
phosphorylation. B, A875 human melanoma cells and
PC12nnr5 cells were infected with adenovirus encoding for LacZ, p75ICD
(ICD), or p75mICD (mICD) at m.o.i. = 25 for
24 h and then treated for 30 min with wortmannin (100 nM) or LY294002 (20 µM) prior to lysis and
analysis by immunoblotting for Akt levels and Akt Ser473
phosphorylation. Results shown were repeated three times with similar
results.
View larger version (51K):
[in a new window]
Fig. 7.
Tyrosine phosphorylation of Shc and p85 is
increased by p75NTR expression. A, PC12nnr5 cells were
infected with LacZ, p75NTR, or p75ICD (ICD) adenovirus for
24 h and, where indicated, incubated in 1 mM sodium
orthovanadate for 1 h and harvested. Shc was immunoprecipitated
(IP), and immunoblots were performed to determine Shc levels
and Shc phosphotyrosine (pTyr) content. B, the
PI3K p85 subunit was immunoprecipitated, and immunoblots were performed
to determine p85 tyrosine phosphorylation and protein levels.
GFP, green fluorescent protein; pS473,
phospho-Ser473. C, PC12nnr5 cells were infected
with LacZ or p75ICD adenovirus for 24 h and, where indicated,
incubated in 1 mM sodium orthovanadate. Cells were lysed,
and glycoproteins were precipitated using wheat germ agglutinin
conjugated to Sepharose. Phosphotyrosine content of the precipitates
was analyzed by 4G10 immunoblotting. As a control, the last
lane shows activated TrkA to emphasize the difference in
SDS-polyacrylamide gel electrophoretic migration of the two proteins.
The phosphorylation state of Akt in the total protein lysates is shown
in the lower panel. These experiments were repeated four
times with similar results. DMSO, dimethyl sulfoxide.
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Fig. 8.
p75NTR expression decreases cytosolic
protein-tyrosine phosphatase activity. PC12nnr5 cells were
infected with LacZ, p75NTR, or p75ICD adenovirus for 24 h and then
harvested in high salt buffer. Cytosolic and membrane compartments were
separated by centrifugation and analyzed for phosphatase activity as
described under "Experimental Procedures." Conditions that were
statistically different from controls (p < 0.001) are
indicated by asterisks. Data shown represent the means ± S.E. of three independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation in most systems (23, 29,
30). Paradoxically, some studies suggest that the receptor signals
apoptosis when free of ligand and that this function is suppressed by
ligand binding to p75NTR (64, 65). For our studies, we produced a group
of recombinant adenovirus that would constitutively activate p75NTR
signaling and thereby allow us to identify p75NTR cascades irrespective
of ligand binding. All of the p75NTR constructs employed specifically
activate the JNK pathway and mediate apoptosis when expressed at high
levels, indicating that they are capable of activating p75NTR signaling
pathways. The high expression levels required to induce apoptosis
presumably reflect forced formation of a receptor signaling complex
normally obtained in the presence of appropriate ligand (46). It is
noteworthy that proximity of the intracellular domain to the plasma
membrane appears important for activation of apoptosis by p75NTR since the p75mICD fragment elicits stronger apoptotic signaling than either
p75NTR or p75ICD.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Farid Arab Said and Sandra MacPherson for technical assistance with adenoviruses; Alain Boudreault for providing phosphatase assay reagents; Jane McGlade and David Kaplan for providing the anti-Shc and anti-TrkA antibodies, respectively; and Wayne Sossin and Sylvie La Boissière for careful reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by operating grants from the Canadian Institutes of Health Research (to P. A. B.).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.
Supported by the Neuroscience Foundation of Canada and by a
Canadian Institutes of Health Research studentship.
§ Supported by a Canadian Institutes of Health Research studentship.
¶ Canadian Institutes of Health Research Scholar.
Killam Foundation Scholar and Canadian Institutes of Health
Research Scholar. To whom correspondence should be addressed: Center
for Neuronal Survival, Montreal Neurological Inst., McGill University,
3801 University Ave., Montréal, Québec H3A 2B4, Canada.
Tel.: 514-398-3064; Fax: 514-398-1319; E-mail:
mdpb@musica.mcgill.ca.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011520200
2 R. T. Dobrowsky, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: NGF, nerve growth factor; p75NTR, p75 neurotrophin receptor; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; TNFR, tumor necrosis factor receptor; JNK, c-Jun N-terminal kinase; PTPase, protein-tyrosine phosphatase; BCS, bovine calf serum; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; m.o.i., multiplicity of infection; CREB, cAMP-responsive element-binding protein; MKK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; FAP-1, Fas-associated phosphatase-1; TRAF, tumor necrosis factor receptor-associated factor.
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