Association of the p75 Neurotrophin Receptor with TRAF6*

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-κB 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 factorreceptor-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-κB 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.

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)(4)(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)(8)(9)(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)(16)(17)(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-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.
Increasing evidence has demonstrated that many TNF receptor family members interact with TNF receptor-associated factors (TRAFs) to modulate JNK and NF-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.
Cell Culture and Transfection-All cells were cultured at 37°C in 5% CO 2 . 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 ϫ 10 6 cells in 10-cm tissue culture dishes were transfected with 10 g 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 ϫ 10 6 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 g/ml aprotinin, 1 g/ml leupeptin, and 25 g/ml phenylmethylsulfonyl fluoride). Lysates (800 g/ml) were then incubated with ␣-FLAG (2 g/ml; Eastman Kodak Co.) and Sepharoseprotein A (Sigma) or with ␣-FLAG M2 Affinity Gel (4 g/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 IgGhorseradish 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).
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-Dlysine 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 g 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 ␣-p65 (1:100, Santa Cruz) and ␣-FLAG (1 g/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 40ϫ objective and quantitated.

RESULTS AND DISCUSSION
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 liganddependent. 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.
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 FIG. 1. TRAF6 associates with p75 in a ligand-dependent manner. A, 293T cells were co-transfected with 5 g 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 g 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 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.
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 immunoblot-

FIG. 3. Expression of endogenous TRAF6 in cultured Schwann cells.
Lysates were prepared from 293T cells, 293T transfected with the FLAG-TRAF6 expression vector, and native Schwann cells prepared from sciatic nerve of P2 rats. Each lysate (50 g) was subjected to gel electrophoresis and Western blotting (WB) with antiserum against TRAF6 (Tularik).

FIG. 4. Effect of DN TRAF6 upon NGF-dependent NF-B translocation in Schwann cells.
Primary cultures of Schwann cells were isolated from P2 rats and plated on coverslips coated with poly-D-lysine. 5 h after plating, the cells were transfected with FLAG-tagged dominant negative DN-TRAF6 cDNA for 6 h using the Effectene method. After overnight incubation in Dulbecco's modified Eagle's medium and 1% fetal calf serum, the cells were serum-starved for 2 h, and 100 ng/ml of NGF was added for 2 h. Cells were then fixed and stained for dominant negative TRAF6 and p65 expression using anti-FLAG and anti-p65 antibody (Santa Cruz), respectively. In addition, cells were stained with 4,6-diamidino-2 phenylinodole as described under "Materials and Methods." A, staining for the NF-B subunit p65 in native Schwann cells. The majority of cells display immunofluorescence for p65 in the cytoplasm. In the presence of NGF (100 ng/ml) for 2 h, positive nuclear staining for p65 was observed. B, transfection of FLAGtagged DN-TRAF6 cDNA blocks translocation of p65. Expression of the dominant negative form of TRAF6 was visualized by anti-FLAG immunocytochemistry. The localization of p65 was determined by immunocytochemistry with anti-p65 antibody. C, quantitation of p65 translocation. Schwann cells were viewed at 40ϫ and quantified for p65 nuclear staining under each condition. Cells that displayed intense nuclear staining over cytoplasmic staining were scored as positive. A fraction of the transfected cells (10%) did not give conclusive staining and were not quantitated in this analysis. FIG. 2. Mapping the interaction between p75 and TRAF6. A, schematic diagram of HA-tagged p75 intracellular deletions. TM represents the transmembrane domain. DD represents the cytoplasmic death domain. B, FLAG-TRAF6 was co-transfected with HA-tagged p75 deletion constructs in 293T cells. Cell lysates were prepared, immunoprecipitated (IP) with an anti-FLAG antibody (Kodak) linked to Sepharose beads. Proteins were separated on a 7.5% polyacrylamide-SDS gel and probed with an anti-HA antibody to detect co-precipitated p75 receptors (top panel). Cell lysates were probed with an anti-FLAG or an anti-HA antibody to follow the expression levels of TRAF6 and p75 receptors, respectively. WB, Western blot. ting 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 NGFmediated NF-B signaling in Schwann cells.
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.
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.