From the Molecular Neurobiology Program, Skirball Institute, New York University Medical Center, 540 New York, New York 10016
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ABSTRACT |
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In addition to the Trk tyrosine kinase receptors,
neurotrophins also bind to a second receptor, p75, a member of the
tumor necrosis factor receptor superfamily. Several signaling pathways have been implicated for p75 in the absence of Trk receptors, including
induction of NF- The neurotrophins use a two-receptor system to promote cell
differentiation and survival, as well as modulate axonal and dendritric branching and synaptic transmission (1, 2).
NGF,1 BDNF and NT-4/5, and
NT-3 bind to TrkA, TrkB, and TrkC, respectively, and also bind to the
p75 receptor (3-5), a member of the TNF receptor superfamily (6). The
TNF family includes the two TNF receptors, Fas-CD95, DR-3, DR-4, CAR-1,
and the lymphoid cell-specific receptors CD30, CD40, and CD27 (7-10).
The common motif that defines these transmembrane proteins is a
40-amino acid cysteine sequence that occurs two to six times in the
extracellular domain. In addition, several members contain a region of
weak homology in the intracellular portion, designated the death domain
(11, 12). Recent NMR analysis has confirmed the existence of a death
domain in the p75 receptor (13).
In the presence of TrkA receptors, p75 participates in the formation of
high affinity binding sites and enhanced neurotrophin responsiveness
leading to increased cell survival (14). In the absence of TrkA
receptors, p75 can generate, in specific cell populations and
conditions, a death signal (15-18). This dichotomy in responses has
raised questions regarding the cell specificity and the nature of the
signaling mechanisms for the p75 receptor.
A variety of signaling pathways has been detected for p75. A transient
elevation in intracellular ceramide levels due to increased sphingomyelin hydrolysis has been observed in several cell types, including T9, NIH3T3, and PC12 cells (19). It is not yet clear what
biological functions are mediated by an increase in ceramide. In
addition, activation of c-Jun N-terminal kinase (JNK) has been observed
in cells undergoing cell death (17, 18, 20). The activity of the
stress-activated JNK protein kinase can be up-regulated by
neurotrophins under apoptotic conditions through p75. Another signaling
response is the activation of the nuclear transcription factor NF- Increasing evidence has demonstrated that many TNF receptor family
members interact with TNF receptor-associated factors (TRAFs) to
modulate JNK and NF- Preparation of the HA-tagged p75 Deletion Constructs--
The 3'
deletions were generated by using polymerase chain reaction (PCR). A
common 5'-PCR primer (5'-GGATATGGTGACCACTGTGATG-3') was used in
conjunction with unique 3'-PCR primers for each particular deletion
( Preparation of the DN TRAF6 Construct--
The DN TRAF6 was
generated using PCR to amplify the TRAF domain. Using 5'
(5'-CGGAATTCCATCTCAGAGGTCCGGAATTTC-3') and 3'
(5'-GAAGATCTCTATACCCCTGCATCAGTACTTCG-3') primers and the pRK5-hTRAF6
cDNA construct as the template (kindly provided by Zhaodan Cao,
Tularik), the PCR product was digested with EcoRI and
BglII and ligated into a EcoRI- and
BglII-digested pCMV2-FLAG cDNA.
Cell Culture and Transfection--
All cells were cultured at
37 °C in 5% CO2. 293T cells were cultured in
Dulbecco's modified Eagle's medium plus 10% fetal calf serum
supplemented with penicillin-streptomycin (Life Technologies, Inc.).
Approximately 1.5 × 106 cells in 10-cm tissue culture
dishes were transfected with 10 µg of plasmid DNA using the calcium
phosphate method.
Immunoprecipitaton and Western Blot Analysis--
Transfected
cells were collected 36 h after removal of calcium phosphate
precipitate and aliquoted into microcentrifuge tubes at 2.0 × 106 cells/ml. Recombinant neurotrophins (provided by
Genentech) were added for various times, as indicated in the text. The
cells were then spun down and lysed in 1 ml of Nonidet P-40 lysis
buffer (1% Nonidet P-40, 20 mM Tris, pH 8.0, 200 mM NaCl, 1 mM EDTA, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 25 µg/ml phenylmethylsulfonyl fluoride).
Lysates (800 µg/ml) were then incubated with Isolation and Transfection of Schwann Cells--
Sciatic nerves
were dissected from P2 rats, cut in small pieces, and incubated in
Hanks' balanced solution containing 0.25% trypsin (Sigma) and 0.25%
collagenase (Sigma) for 30 min. The cells are triturated, plated on
poly-D-lysine coated coverslips, and cultured in
Dulbecco's modified Eagle's medium containing 1% fetal calf serum
and glial growth factor (63 ng/ml; Cambridge Neuroscience). Schwann
cells are transfected with 0.3 µg of cDNA using the Effectene
Transfection Reagent kit (Qiagen). After transfection, the cells were
serum-starved for 2 h, and then 100 ng/ml of NGF was added for an
additional 2 h. The cells are fixed in 4% paraformaldehyde, blocked in 5% goat serum, and incubated with The TRAF6 protein interacts with the B cell antigen CD40 through a
homology domain at the C-terminal region, the TRAF domain (25). TRAF6
also contains an N-terminal RING finger sequence, five zinc fingers,
and a coiled-coil domain that are required for TRAF6 signaling. To
determine whether TRAF6 interacts with p75, 293T cells were
cotransfected with p75 cDNA and TRAF6, tagged with the FLAG epitope
(FLAG-TRAF6). Immunoprecipitation of lysates with antibodies against
FLAG, followed by immunoblotting with p75 antibodies indicated that p75
associated with TRAF6 (Fig. 1A). In the absence of TRAF6
expression, immunoprecipitation of p75 was not observed. The
association of TRAF6 with p75 was ligand-dependent. Indeed,
addition of increasing doses of NGF enhanced the interaction (Fig.
1B). The recruitment of TRAF6 to the receptor in cells
treated with NGF was rapid. Within 1 min after NGF treatment, an
increase level of association was detected (Fig. 1C). TRAF6
migrated as a protein with a molecular mass of 60 kDa. In these
experiments an equal amount of TRAF6 protein was detected in each
immunoprecipitation reaction. These results indicate that TRAF6
associates with p75 in a ligand-dependent manner in 293T
cells.
B and c-Jun kinase activities and increased production of ceramide. However, to date, the mechanisms by which the
p75 receptor initiates intracellular signal transduction have not been
defined. Here we report a specific interaction between p75 and TRAF6
(tumor necrosis factor
receptor-associated factor-6) after
transient transfection in HEK293T cells. The interaction was
ligand-dependent and maximal at 100 ng/ml of nerve growth factor (NGF). Other neurotrophins also promoted the association of
TRAF6 with p75 but to a lesser extent. The binding of TRAF6 was
localized to the juxtamembrane region of p75 by co-immunoprecipitation and Western blotting. To assess the functional significance of this
interaction, we have tested responses in cultured Schwann cells that
express p75 and TRAF6. An NGF-mediated increase in the nuclear
localization of the p65 subunit of NF-
B could be blocked by the
introduction of a dominant negative form of TRAF6 in Schwann cells.
These results indicate that TRAF6 can potentially function as a signal
transducer for NGF actions through the p75 receptor.
INTRODUCTION
Top
Abstract
Introduction
References
B.
It has been shown that NF-
B activity can be induced by NGF in
primary Schwann cells expressing p75 but not TrkA receptors (21). This
stimulation was not observed by BDNF or NT-3 treatment of Schwann
cells, which express TrkB and TrkC.
B activity as well as apoptosis (22, 23). So far
six TRAF proteins have been identified that mediate signaling through
receptors for TNF, CD30L, CD40L, and IL-1 (24). TRAF-6 is expressed in
neural tissues (25), leading us to test whether this TRAF protein was
capable of associating with the p75 neurotrophin receptor. Here we
report a specific interaction of the p75 neurotrophin receptor with
TRAF6. This association leads to an translocation of the p65 subunit of
NF-
B in Schwann cells.
MATERIALS AND METHODS
35, 5'-ATAAGGGCCCTCAGCGTCGCAGGGCGGCTAAAAG-3';
62, 5'-ATAAGGGCCCTCACTCGCCTGCCAGATGTCGC-3';
86,
5'-ATAAGGGCCCTCACAGGGGCAGGCTACTGTAGAG-3';
113,
5'-ATAAGGGCCCTCAGCTGTCCACAGAGATGCCAC-3'; and
128,
5'-ATAAGGGCCCTCACGGTGGGGGCGTCTGGTTCAC-3'), and the pCDNA3-rat p75
cDNA was used as a template. All PCR products were cut with
NarI and ApaI and ligated into NarI-
and ApaI-digested pCDNA3-p75 carrying the rat p75
cDNA sequence. The 5' HA epitope was generated by PCR using 5'
(5'-GGGGTACCACCATGTCTGCACTTCTGATCCTAGCTCTTGTTGGAGCTGCAGTTGCTTATCCATATGATGTTCCAGATTATCTAAGGAGACATGTTCCACAG-3') and 3' (5'-CTTGGGATCCATCACCAGG-3') primers with the
pCDNA3-p75 cDNA as the template. The PCR product was digested
with KpnI and BamHI and ligated directly into
KpnI- and BamHI-digested pCDNA3-p75 deletion
cDNAs. All constructs were verified by DNA sequencing (Rockefeller
University, New York, NY).
-FLAG (2 µg/ml;
Eastman Kodak Co.) and Sepharose-protein A (Sigma) or with
-FLAG M2
Affinity Gel (4 µg/ml; Kodak). The matrix was then washed, and immune
complexes were subjected to Western analysis. Samples in
SDS-polyacrylamide gel electrophoresis sample buffer were resolved on a
10% SDS-polyacrylamide gel under reducing conditions. Proteins were
transferred to polyvinylidene difluoride membrane, blocked with 5%
milk, and incubated with a primary rabbit polyclonal antibody for p75,
9992, at 1:5000 or a rabbit polyclonal
-HA antibody (Immunotech) at
1:1000 dilution at room temperature. A polyclonal antibody to TRAF6 was
provided by Zhaodan Cao (Tularik) and used at a dilution of 1:500. The
membrane was washed with Tris-buffered saline and Tween and incubated
with an
-mouse IgG-horseradish peroxidase (Sigma) and then processed
by ECL (Amersham Pharmacia Biotech) and exposed to x-ray film. The gels
were scanned and quantitated by ImageQuant v1.1 (Molecular Dynamics).
-p65 (1:100, Santa Cruz) and
-FLAG (1 µg/ml) overnight at 4 °C. The cells are then incubated with biotinylated anti-rabbit IgG (1:500, Vector) for 1 h, followed by incubation with rhodamine avidin D cell sorting (1:200,
Vector), goat anti-mouse fluorescein isothiocyanate (1:200, Jackson
Laboratories), and 4,6-diamidino-2 phenylinodole (1:500) for 1 h.
The coverslips were mounted using Vecta Shield. Cells were viewed at
with a 40× objective and quantitated.
RESULTS AND DISCUSSION
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Fig. 1.
TRAF6 associates with p75 in a
ligand-dependent manner. A, 293T cells were
co-transfected with 5 µg each of pCDNA3-p75 and either FLAG-TRAF6
or vector control, pFLAG-CMV2. Cells were collected 36 h after
transfection, pooled, and divided evenly. NGF (100 ng/ml) was added for
5 min before the cells were lysed in 1% Nonidet P-40 lysis buffer. The
lysates were immunoprecipitated with anti-FLAG antibody and separated
on a 10% polyacrylamide-SDS gel. After transfer, the proteins were
analyzed for p75 using an antibody directed against the cytoplasmic
domain of the receptor (9992, top panel). The immunoblot is
stripped and reprobed for relative levels of immunoprecipitated
(IP) FLAG-TRAF6 (bottom panel). The levels of p75
receptor and TRAF6 following immunoprecipitation were quantitated and
expressed as a ratio of p75 to TRAF6 immunoreactive protein. The
quantitation is presented under each gel. The ratio was represented
relative to the signal derived from transfected cells in the absence of
neurotrophin. B, 293T cells were co-transfected as described
above. NGF was added to the cells at the specified concentrations for 5 min. The lysates were then immunoprecipitated with anti-FLAG antibody
and subjected to Western blotting for p75. C, 293T cells
were co-transfected with 5 µg of pCDNA3-p75 and FLAG-TRAF6 and
treated with 100 ng/ml of NGF. Lysates were prepared and subjected to
immunoprecipitation and Western blot analysis. D, effect of
neurotrophins upon TRAF6 interaction with p75. 293T cells were
transfected with 5 µg of pCDNA3-p75 and FLAG-TRAF6. Cells were
then treated with 100 ng/ml of NGF, BDNF, NT-3, or NT-4/5 for 5 min.
The cells were lysed and subjected to immunoprecipitation of TRAF6
followed by Western blotting with anti-p75 antibodies. represents no
neurotrophin added to the cells. Equal expression of p75 and TRAF6 was
confirmed by Western analysis.
The p75 receptor binds equally well to NGF, BDNF, NT-3, and NT-4/5. We therefore tested whether other neurotrophins could promote the association of TRAF6 and p75. Addition of NGF or NT-4/5 to transfected 293T cells for 5 min resulted in approximately 5-fold increase of p75 associated with TRAF6 (Fig. 1D). However, treatment with 100 ng/ml of BDNF and NT-3 produced a smaller degree of association between p75 and TRAF6 (2- and 1.5-fold, respectively). Similar amounts of TRAF6 (Fig. 1D, lower panel) and p75 were present in each immunoprecipitation. This experiment was repeated with a similar outcome. Although each neurotrophin binds to the p75 receptor with a similar affinity (27, 28), several p75 specific functions are preferentially activated by NGF and not by BDNF or NT-3 (17, 21). The results here indicate that each neurotrophin promotes differential interactions between p75 and TRAF6.
To map the interaction between p75 and TRAF6, a series of deletion
mutants was made in the cytoplasmic domain of p75 (Fig. 2A). The intracellular domain
of p75 is 154 amino acids in length and contains a highly conserved
juxtamembrane region and a death domain sequence in the C terminus
(13). To facilitate detection of these constructs, each p75 deletion
was epitope-tagged with HA at the N terminus. The p75 receptor
deletions were transiently cotransfected with FLAG-TRAF6 in 293T cells.
Lysates were immunoprecipitated with anti-FLAG antibody and
immunoblotted with an anti-HA antibody to detect the p75 receptor
deletions. TRAF6 was found to interact with p75 deletion 35,
62,
83, and
113 but not a deletion missing the distal 128 amino
acids,
128 (Fig. 2B). Analysis of lysates that expressed
the p75 receptor alone (HA-p75) or TRAF6 alone did not produce
immunoprecipitation of either protein (Fig. 2B). Comparable
expression of TRAF6 and p75 receptors was confirmed by Western blotting
(Fig. 2B, lower panel). This analysis indicated that the interaction between p75 and TRAF6 occurred in the
juxtamembrane region of p75, requiring sequences between residues 113 and 128.
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What is the physiological significance of an association between p75
and TRAF6? Previous studies indicated that NGF may promote migration
and L1 cell adhesion protein expression in Schwann cells (29, 30). A
potential signaling pathway to account for these activities may be
NF-B activation, previously shown to be dependent upon NGF binding
to p75 (21). To test whether TRAF6 is expressed in Schwann cells, we
probed lysates prepared from cultured Schwann cells by immunoblotting
with antisera directed against TRAF6. The TRAF6 protein of 60 kDa was
detected in both Schwann cells and 293T cells. As an additional
control, transfection of 293T cells with the FLAG-TRAF6 protein lead to
overexpression of the protein (Fig. 3).
Introduction of a TRAF6 cDNA construct with an NF-
B luciferase
reporter construct in Schwann cells lead to a 5-fold increase in
luciferase activity (data not shown). These results suggest that TRAF6
may be a component of NGF-mediated NF-
B signaling in Schwann
cells.
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To assess whether TRAF6 is involved in NGF signaling, we utilized a DN
form of TRAF6 that contains the C-terminal half of TRAF6 (amino acids
289-522). This form of TRAF6 was used previously to show that TRAF6
was required for IL-1 and CD40 mediated NF-B activation (25, 31).
Translocation of the p65 subunit of NF-
B to the nucleus was used as
an assay. Freshly dissected Schwann cells were transfected with the
DN-FLAG-TRAF6, fixed, and subjected to indirect immunofluoresence with
antibodies against p65 and the FLAG epitope. In the absence of NGF
treatment, Schwann cells displayed staining for p65 predominately in
the cytoplasm (Fig. 4A).
Addition of NGF resulted in strong redistribution of p65 immunoreactivity to the nucleus. On the other hand, Schwann cells expressing DN-TRAF6 did not show a redistribution of nuclear staining for p65 in the presence of NGF (Fig. 4B). The expression of
DN-TRAF6 was monitored by FLAG antibody staining.
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To quantitate the effect of DN-TRAF6 in Schwann cells, the percentage
of cells exhibiting p65 nuclear staining was measured. In the absence
of NGF, a fraction of the nontransfected cells (20%) stained positive
for p65 staining. The addition of NGF resulted in 60% of the Schwann
cell population being positive for p65 nuclear staining (Fig.
4C). Cells expressing DN-TRAF6 did not display a significant
increase in p65 nuclear staining. These results verify that NGF can
promote NF-B nuclear translocation in Schwann cells. It should be
noted that the translocation of p65, as well as increases in NF-
B
activity, was not observed in Schwann cells grown for more than 1 week
(data not shown), indicating that Schwann cells lose responsiveness
over time in culture. Nevertheless, expression of DN-TRAF6 inhibited
p75-dependent NF-
B translocation in freshly dissociated
cells. Hence, TRAF6 is a potential mediator of
NGF-dependent NF-
B activation through the p75 receptor.
Although p75 has not been generally regarded as a signal transducing receptor, recent evidence indicates that it can signal independently in certain cellular contexts (32, 33). The phenotype of the p75 null mice supports a role in neuronal survival (34, 35) and suggests an apoptotic function as well (36, 37). Another paradoxical issue regarding p75 action that has been raised is that although all neurotrophins (NGF, BDNF, and NT-3) bind to p75 with similar affinity, each neurotrophin may exert different effects on cell function and viability through p75 (4, 16, 17, 21). One explanation to account for the effect of distinct neurotrophins is the co-expression of Trk receptors. Differences in the kinetics of binding and the degree of positive cooperativity of each neurotrophin to p75 (27, 28) may produce different outcomes. Finally, recruitment of adaptor molecules by p75 may be different with each neurotrophin. The finding that NGF and NT-4/5 produce a stronger TRAF6 association with p75 than BDNF and NT-3 is consistent with this conclusion.
The deletion analysis localized the binding of TRAF6 to a highly
conserved region of the p75 cytoplasmic region, which includes the
amino acids EGEKLHSDSGISVDS. This juxtamembrane sequence has been
completely conserved between human, rat, and chicken p75 genes (38).
TRAF6 can also be recruited to the IL-1 receptor through binding to
IRAK (IL-1 receptor-associated
serine-threonine kinase) (31, 39). Although the binding
domain of IRAK for TRAF6 has not been defined, a comparison of IRAK to
the cytoplasmic domains of p75 and CD40 did not yield a common
consensus binding sequence. Therefore, the interaction of TRAF6 with
other signaling partners appears to require different binding
sequences. In addition, downstream signaling through TRAF6 can take
place through the NF-B-inducing kinase (NIK), a mitogen-activated
protein kinase kinase kinase member (40), or through the apoptosis
signal-regulating kinase, ASK1 (41). Further studies are required to
establish whether these enzymes are also required for neurotrophin signaling.
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ACKNOWLEDGEMENTS |
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We thank Bruce Carter, Hiroko Yano, Yong Won Choi, Craig Thompson, and Donna Osterhout for advice and Zhaodan Cao (Tularik), Mark Marchionni (Cambridge Neuroscience), and H. C. Liou for reagents.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health and the American Heart Association.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: Molecular Neurobiology
Program, Skirball Inst., New York University Medical Center, 540 First
Ave., New York, NY 10016.
The abbreviations used are: NGF, nerve growth factor; TNF, tumor necrosis factor; TRAF, tumor necrosis factor receptor-associated factor; BDNF, brain-derived neurotrophin factor; NT, neurotrophin; IL, interleukin; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; PCR, polymerase chain reaction; DN, dominant negative.
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REFERENCES |
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