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Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is activated by Tumor Necrosis Factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) upon TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adaptor molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose dependent manner, upon TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2 dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.
Mixed Lineage Kinase 3 (MLK3) is a MAPKKK and belongs to MLK family . The MLK family members are characterized by the presence of signature sequences of Serine/Threonine (Ser/Thr) and Tyrosine kinases within their catalytic domain. We and others have shown that some of the MLK members, including MLK3 , MLK2  and DLK  possess the functional Ser/Thr kinase activities, however, the tyrosine kinase activity of any MLK family member is still not known. Structurally MLK family members are highly conserved in their catalytic domain but they diverge in the C-terminal domain, suggesting that probably each family member might be regulated differentially by different ligands. Although the specific ligands for all MLK family members are not yet known, we reported earlier that MLK3 can be activated by TNF-α . The TNF-α was also able to activate JNK, the downstream target of MLK3, specifically in a MLK3-dependent manner, where as the activation of other two MAPKs, p38 and ERK was not mediated via MLK3 . Although our studies showed that TNF-α is a ligand of MLK3 , however the mechanism by which TNF-α activates MLK3 is still not understood.
TNF-α is a pleiotropic cytokine that renders cellular effects by binding to its cognate receptors, TNFR1 and TNFR2. It has been suggested that upon encounter with trimeric TNF-α, the TNF receptors undergo trimerization, which leads to intracellular signaling [6, 7]. Recently, it has been reported that TNFR proteins self assemble in the absence of ligand, undergoing conformational changes upon ligand engagement that lead to downstream signaling . Upon conformational changes in the TNFRs, the cytoplasmic ends of TNFRs bind to specific adaptor proteins that ultimately transmit the signals to downstream targets . Since TNFR proteins lack intrinsic enzymatic activity, the binding of adaptor proteins to the cytoplasmic end of the receptor was shown to be essential for TNF-α mediated signaling . One of the protein families that bind to both the TNFRs is TRAFs that has been shown to activate JNK and NF-κB pathways upon TNF-α stimulation .
Till date, six members of the TRAF family have been described [11-13]. All the TRAFs contain C-terminal TRAF domain that mediates the interaction with TNF receptors and hetero- or homo- dimerization among the TRAF family members . In addition to the TRAF domain, the N-terminus of the TRAF proteins, with the exception of TRAF1, contains RING domain and multiple Zinc finger structures; those are essential for their effectors functions [9, 12]. The TRAF family not only mediates TNF receptor family-mediated signaling but may also link the downstream signaling via other receptor like interleukin-1 receptor (IL-1R) . Upon IL-1R receptor engagement, the cytoplasmic end of the receptor recruits a protein complex, including IRAK that binds with TRAF6 and activates JNK, p38, ERK and Src tyrosine kinase [13-16]. Among the six known TRAF proteins, TRAF2, TRAF5 and TRAF6 are reported to activate JNK , the downstream target of MLK3  and other MLKs . It is also reported that TRAF proteins activate the MAPK cascades at the level of MAP3K and MAP4K, including ASK1 [18, 19], TAK1 , MEKK1 , NIK  and GCK . Whether, the TRAF proteins also recruit MLK3 for TNFR-mediated activation of downstream JNK is yet to be determined.
In this report, we show that although TRAF2, TRAF5 and TRAF6 can all bind to MLK3, however, only TRAF2 can activate the MLK3 kinase activity. The interaction between TRAF2 and MLK3 was mediated via the TRAF domain of TRAF2, and the C-terminal domain of MLK3. The endogenous TRAF2 and MLK3 also associate with each other, in response to TNF-α treatment and TRAF2 associated MLK3 possessed significantly higher kinase activity compared to total MLK3 pool. The association between TRAF2 and MLK3 was essential for TNF-α-induced MLK3 activation, since competition with the TRAF domain of TRAF2 was able to attenuate MLK3 activation in a dose-dependent manner. We also show that TRAF2 was essential for TNF-α-induced JNK activation in a MLK3-dependent manner. Together, these data show a role of TRAF2 in TNF-α-induced MLK3 activation, and it’s downstream signaling to JNK pathway.
To determine whether TRAF proteins play any role in the activation of MLK3 via TNFRs, we first attempted to identify the TRAF protein(s) that binds with ectopically expressed MLK3. The human HEK-293 cells were transiently transfected with equal amounts of GST-tagged MLK3 along with either Flag-tagged or Myc-tagged TRAF expression vectors, as indicated (Fig. 1). The TRAF proteins were immunoprecipitated either by anti-FLAG (for TRAF2, 5 and 6) or by anti-Myc (for TRAF1 and 3) antibodies and any associated recombinant GST-tagged MLK3 was detected by anti-GST antibody. Our results showed that TRAF2, 5 and 6 associated with ectopically expressed MLK3, however TRAF1 and 3 failed to co-immunoprecipitate with MLK3 (Fig.1). Taken together, these results indicate that MLK3 associates with TRAF2, 5 and 6 under over expressed condition.
Interaction between TRAF2 and some MAP3K members, such as ASK1 [18, 19], TAK1  and MEKK1  has been shown to induce their kinase activities. We examined which of the TRAF proteins (i.e. TRAF2, 5 and 6) that showed interaction with MLK3 (Fig.1) in fact can activate MLK3 kinase activity. HEK-293 cells were transiently transfected with a constant amount of MLK3 and the three TRAF expression vectors, as shown in Fig. 2. As controls, some cells were also transfected with TRAF expression vectors alone, to rule out the contribution of any contaminating kinase during the kinase assay. Recombinant MLK3 was precipitated by GSH-pull down and subjected to kinase assay using MLK3 specific substrate, SEK1 (K-R) protein, expressed in bacteria. MLK3 was about 3.5 fold more active in the cells, where TRAF2 was transfected (Fig. 2A, compare lanes 2 with 4) where as MLK3 was not significantly activated in cells that were transfected with TRAF5 and 6 (Fig. 2A). The expression levels of MLK3 and TRAFs were almost equal in all our transfection experiments. These results suggest that TRAF2 might be the adaptor protein that recruits MLK3 to the TNFR. To further confirm that our results were not due to transfection artifact, mouse embryonic fibroblasts (MEFs) from wild type and TRAF2 deficient mice were utilized to examine whether TRAF2 is required for TNF-α-induced MLK3 activation. The wild type (TRAF2+/+) and TRAF2 deficient (TRAF2-/-) MEFs were treated with 10nM of TNF-α for 30 or 60 minutes and the endogenous MLK3 kinase activity was measured. Endogenous MLK3 was activated about 4.5 fold following 30 or 60 minutes stimulation by TNF-α in wild type and not in TRAF2 deficient MEFs (Fig. 2B, compare lanes 4 and 5 with 8 and 9). The time period for TNF-α treatment was chosen based on our results as shown in Fig. 3. Taken together these results conclusively demonstrate that TRAF2 is necessary for TNF-α mediated MLK3 activation.
Given that TRAF2 protein can associate (Fig.1) with and activate ectopically expressed MLK3 (Fig. 2A), we planned to test the association between TRAF2 and MLK3 under more physiological condition. The Jurkat T cells were chosen for these experiments, since these cells respond very well to TNF-α treatment and express considerable amounts of endogenous MLK3. To examine the endogenous interaction between MLK3 and TRAF2, Jurkat T cells were starved in low serum (0.2% FBS) containing medium for 12 hours and then treated with human TNF-α for different period of times. The endogenous TRAF2 was immunoprecipitated and any associated MLK3 in the immune-complex was blotted with antibody against MLK3. Endogenous MLK3 co-immunoprecipitated with TRAF2 in a time dependent manner and the interaction was maximal at 30 minutes of TNF-α treatment and was undetectable by 120 minutes (Fig. 3A). To further confirm that the endogenous interaction between MLK3-TRAF2 was not due to any artifacts of non-specific interaction, MEFs from wild type and TRAF2 deficient mice were treated with murine TNF-α, similar to Jurkat T cells. Endogenous TRAF2 was immunoprecipitated and blotted for any associated MLK3, similar to Jurkat T cells. The association between endogenous TRAF2 and MLK3 was only observed in TRAF2 immunoprecipitates from wild type MEFs, but not in TRAF2 deficient MEFs in response to TNF-α treatment (Fig. 3B). This association between MLK3 and TRAF2 was also higher in wild type MEFs at 30 minutes of TNF-α treatment (Fig. 3B), as observed in Jurkat T cells. Therefore, it can be concluded that the interaction between MLK3 and TRAF2 is physiological and is regulated by TNF-α.
The sequence analysis of TRAF family proteins reveal several conserved domains, such as RING, Zinc finger and TRAF domains, which are reported to mediate protein-protein interactions. To identify the domain(s) on TRAF2 proteins that interact with MLK3, several TRAF2 deletion constructs were utilized, as shown in Fig. 4A. These TRAF2 mutant constructs were transfected into HEK-293 cells along with MLK3 expression vector, as indicated in Fig. 4B. First, the expression levels of all TRAF2 mutants, including the full length proteins were analyzed, and then the cell lysates with equal expression of TRAF2 mutant proteins were immunoprecipitated using anti-FLAG antibody. The association between MLK3 and TRAF2 mutant proteins was detected using an antibody against the GST-tag present on recombinant MLK3. As per our expectation, MLK3 interacted with full length and other TRAF2 mutant proteins, except the TRAF2 mutant protein, where the entire TRAF domain was deleted (Fig. 4B, lane 4). Interestingly, the interaction between entire TRAF domain and MLK3 was the least (Fig. 4B, lane 5) whereas it was highest with the N-terminus of the TRAF domain (Fig. 4B, lane 7). Our result is consistent with one other protein, where it has been reported that isolated TRAF domain also does not interact with ASK1; even though the deletion of TRAF domain from TRAF2 fails to interact with full length ASK1 . However, our results, clearly suggest that TRAF2 interact with MLK3 via TRAF-N domain (Fig. 4B).
Similar to TRAF2, MLK3 also contains several conserved domains, which have been shown to mediate protein-protein interactions . Several deletion mutants (Fig. 4C) of MLK3 proteinwere created to map the specific site on MLK3, required for interaction with TRAF2. These constructs were transfected in HEK-293 cells along with Flag-tagged, full length TRAF2 expression vector. The interaction between TRAF2 and MLK3 mutant proteins were determined by immunoprecipitating TRAF2 by anti-FLAG antibody and blotting with anti-GST antibody for associated MLK3 mutant proteins. As per our earlier observation (Fig. 1), full length MLK3 interacted with TRAF2 and all other mutant proteins, except the C-terminal domain of MLK3 failed to interact with full length TRAF2 (Fig. 4D, lane 10). These results suggest that MLK3 interact with TRAF2 via its C-terminal regulatory domain.
TRAF2 is an adaptor protein and lacks any intrinsic kinase activity and thus we wanted to know how TRAF2 activates MLK3 kinase activity. One potential way by which TRAF2 might activate MLK3 is via protein-protein interaction in response to TNF-α treatment. To test whether direct interaction of TRAF2 with MLK3 activates MLK3 kinase activity by TNF-α, Jurkat T cells were treated with TNF-α for different time points, as indicated in Fig. 5A. First, we optimized the conditions to immunoprecipitate equal amounts of total MLK3 and TRAF2 associated MLK3 by using MLK3 and TRAF2 antibodies respectively (Fig. 5A). Once we optimized how much of cell lysates were needed to immunoprecipitate equal amounts of MLK3 by both the antibodies, we immunoprecipitated comparable amounts of total and TRAF2 associated MLK3 as shown in Fig. 5A, and measured MLK3 kinase activity by using SEK1 (K-R) protein. As envisioned, our results showed about 4 fold activation of TRAF2-associated MLK3 when compared to equal amount of total MLK3 after 30 minutes of TNF-α treatment (Fig. 5A, compare lanes 5 with 6). These results clearly suggest that TNF-α-induced TRAF2-MLK3 interaction is required for MLK3 activation. To further confirm that the interaction between TRAF2-MLK3 indeed is required for induction of MLK3 kinase activity, we attempted to compete off this interaction by using the TRAF-N domain that we determined to be the minimal domain on TRAF2 for MLK3 interaction (Fig. 4B). Constant amount of MLK3 and increasing quantity of cDNA expressing TRAF-N domain (AA, 272-358) were transfected in HEK-293 cells and treated with either 10nM of TNF-α for 30 minutes, or left alone, as controls (Fig. 5B). Equal amount of MLK3 was immunoprecipitated from these cells and subjected to kinase assay. Confirming our endogenous interaction data (Fig. 5A), the MLK3 kinase activity was gradually attenuated/competed off by increasing dose of TRAF-N domain in TNF-α treated cells but not in untreated cells (Fig. 5B). These results clearly suggest that TNF-α induces interaction between TRAF2 and MLK3 and this interaction leads to MLK3 activation.
Earlier we have shown that MLK3 was necessary for TNF-α-induced JNK activation  and MLK3 specifically mediated TNF-α-induced JNK activation and not other MAPKs . To examine whether TRAF2 mediates TNF-α action on JNK via MLK3, we took advantage of TRAF2 deficient MEFs. These cells were transfected with TRAF2 expression plasmids or used without transfection as controls. Two days post-transfection, these cells were treated with TNF-α for 30 or 60 minutes and JNK kinase activity was measured using bacterially expressed GST-c-JUN as the substrate. The basal activity of JNK in these cells was very low upon TNF-α treatment in absence of endogenous TRAF2 (Fig. 6A, lane 2) and was slightly elevated upon ectopic expression of TRAF2, without TNF-α treatment (lane 3). However, JNK activity was increased about 3.5 fold in these cells, when TRAF2 was ectopically expressed, and treated with TNF-α for 30 or 60 minutes (Fig. 6A). The MLK3 kinase activity was also estimated from these cells, which showed activation profile similar to that of JNK (Fig. 6B). Based on these results and our previous published results , we conclude that TRAF2 is necessary for MLK3-mediated JNK activation by TNF-α.
To further confirm that MLK3 is required for TNF-α-induced JNK activation via TRAF2, normal MEFs (i.e. TRAF2+/+) were used to knock down the endogenous MLK3 by transducing these cells with lentivirus, containing MLK3 specific shRNA. Cells transduced with LacZ shRNA were used as controls (Fig. 6C). These cells were treated with murine TNF-α for 30 min or left alone as controls. Western Blot analysis with anti-MLK3 antibody, clearly showed about 95% knock down of endogenous MLK3 by MLK3 specific shRNA (Fig. 6C, lower panel, lane 3 and 4) but not by LacZ shRNA (lanes 5 and 6). Equal amount of cell lysates were then blotted with phospho-JNK antibody to determine the activation status of endogenous JNK in these cells. Our results showed that TNF-α indeed activated endogenous JNK in control cells with endogenous MLK3 (Fig. 6C, upper panel lanes 2 and 6), however the activation of JNK by TNF-α was attenuated significantly when MLK3 was knocked down by MLK3 specific shRNA (see lane 4). Collectively these results convincingly prove that TRAF2-MLK3 pathway mediates the TNF-α-induced JNK activation.
We have shown earlier that MLK3 was activated by TNF-α . In this study, we also reported that TNF-α-induced MLK3 activation, specifically activated JNK but not p38 or ERK MAPKs ,suggesting that TNF-α-induced MLK3 activity was actually specific for JNK activation. Here, we provide a mechanism by which TNF-α activates MLK3 and its downstream target JNK.
The family member of TRAF adaptor proteins have been implicated in cytokine-mediated downstream signaling, where TRAF2, TRAF5 and TRAF6 have been shown to activate the JNK pathway . Our initial expectation was that MLK3 might interact with all three TRAF proteins, which are already implicated in the activation of JNK, which will lead to induction of MLK3 catalytic activity. Our results clearly suggest that all three TRAF proteins (i.e. TRAF2, TRAF5 and TRAF6) can interact with ectopically expressed MLK3 (Fig. 1), but only TRAF2 can activate MLK3 kinase activity (Fig. 2A). It remains a possibility that under specific physiological conditions and in response to specific stimuli, TRAF5 and 6 proteins might also lead to MLK3 activation, which is yet to be determined. The interaction between TRAF2 and MLK3 is physiological based on the fact that endogenous TRAF2 can interact with MLK3 in response to the ligand of MLK3 i.e. TNF-α (Fig. 3), in a time dependent manner.
TRAF2 interaction with other MAP3Ks has previously been shown to cause their activation [18-21], but how the interaction per-se leads to increase in the catalytic activities of these kinases is still not known. We also observed that the interaction between MLK3 and TRAF2 was essential for TNF-α-induced activation of MLK3 because the TRAF2-associated MLK3 was 4.5 fold more active compared to the total cellular pool of MLK3 (Fig. 5A). Furthermore, we also confirmed that the activation of MLK3 by TNF-α was via TRAF2 interaction because MLK3 activation was attenuated in a dose dependent manner by the TRAF-N protein (Fig. 5B) that interacts with MLK3 (Fig. 4A). Thus collectively, we can conclude that TRAF2-MLK3 interaction is essential for MLK3 activation by TNF-α. During review of this manuscript, similar findings were reported by Korchnak et al . We reported earlier that in addition to TNF-α, the bioactive membrane lipid, ceramides also activates MLK3  and therefore one potential mechanism by which TRAF2 interaction with MLK3 can lead to its activation might be via recruiting MLK3 to the membrane. When MLK3 is recruited at the membrane, the ceramides generated via TNF-α-induced sphingomyelinase activation  might modulate MLK3 kinase activation, which is yet to be determined. We also examined any association between MLK3 and TRAF2 by ceramide in Jurkat cells. The ceramide concentration that activated the endogenous MLK3 in Jurkat cells did not induce any interaction between TRAF2 and MLK3 (data not shown), suggesting that mechanisms of MLK3 activation by TNF-α and ceramides are distinct.
TRAF2 has been shown to cause activation of both JNK and NF-κB pathways . Although, now it is known that TRAF2 primarily activates JNK pathway rather than NF-κB, as the MEFs from TRAF2 deficient mice were defective in JNK activation but not in NF-κB pathway activation . One report also implicated MLK3 in the activation of NF-κB pathway in T cells by activating IKKs ; however we were unable to reproduce similar effect of MLK3 on NF-κB activation in T cells. Interestingly other MAP3K members, like ASK1 and MEKK1 are also activated via TNF-α-induced TRAF2 interaction and have been reported to activate their downstream JNK pathway [18, 19, 21, 29]. However, studies in mice deficient for these kinases could not support an essential role of these kinases in TNF-α-induced JNK activation, suggesting some other MAP3K member might be essential for TNF-α-induced JNK activation [30, 31]. Thus, so far, it can be concluded that MLK3 is a primary MAP3K in TNF-JNK pathway, which was also confirmed by an earlier report in MLK3 knockout MEFs . In addition, we also observed that MLK3-induced JNK activation does require TRAF2 (Fig. 6A).
Based on our current data and several published results, we propose a model for JNK and NF-κB pathway activation via TNFRs-TRAF2 pathway. The TNF-α-induced JNK and NF-κB pathways bifurcate at the level of TRAF2, where MLK3 interacts with TRAF2 upon TNF-α stimulation and phosphorylates MKK4 and MKK7 (two MAPKK members in JNK pathway) to phosphorylate and activate downstream JNK that finally translocates to the nucleus and regulate AP1-mediated transcription. Since TRAF2 deficiency purely down regulates JNK and not NF-κB pathway, TNF-α might utilize other TRAFs, such as TRAF5 to activate NIK, which eventually leads to NF-κB activation. In addition, the death domain containing protein RIP is required for TNF-α-induced NF-κB activation. Since we did not observe any NF-κB activation in Jurkat T cells upon MLK3 over expression (data not shown), we can conclude that TNF-α-induced interaction between MLK3 and TRAF2 primarily induce JNK pathway and not NF-κB.
In conclusion, our data provide an insight into the mechanism by which TNF-α activate MLK3. Our biochemical data clearly show that TRAF2-MLK3 interaction is necessary for MLK3 and its downstream target JNK activation. The interaction between MLK3 and TRAF2 is physiological because these proteins interact endogenously upon TNF-α treatment, in a time dependent manner and the activation of MLK3 was specifically blocked by the mutant of TRAF2 that binds with the C-terminus of MLK3 protein. We are currently in the process of determining in details, how the interaction between TRAF2 and MLK3 leads to MLK3 activation.
Human embryonic kidney 293 (HEK293) cells, TRAF2 deficient (TRAF2-/-) and normal Mouse Embryonic Fibroblasts (MEFs) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). Jurkat T cells were maintained in RPMI medium containing 4.5g/L glucose, 10mM HEPES, 1.0mM sodium pyruvate, 10% FBS and 50μM β-ME. For TNF-α treatment, either murine TNF-α (Roche Applied Science, Mannheim Germany) for cell lines of murine origin, or human TNF-α (Roche Applied Science, Mannheim, Germany) for cell lines of human origin, were used, as indicated. The cells were starved in 0.2% serum containing media for 12 hours prior to treatment with 10nM TNF-α for the indicated periods of time.
The Flag-tagged TRAF2 clones were obtained from Dr. James R. Woodgett (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada) as described . Myc-tagged TRAF1 and TRAF3 were obtained from Dr. Bharat Agarwal (MD Anderson Cancer Center, Houston, Texas) and all other TRAFs (i.e. TRAF5 and 6) were from Dr. Woodgett’s laboratory . The MLK3 clones were constructed in our laboratory, as described earlier [24, 33]. For cDNA transfection, in most cases, 1μg of various cDNAs were taken, otherwise as indicated. The various cell types used were transfected with the appropriate expression vectors using LipofectAMINE (Invitrogen, Carlsbad, CA) reagent as per manufacturer instruction. The endogenous MLK3 was knocked down using MLK specific shRNA. The shRNA oligos were synthesized commercially (Invitrogen, Carlsbad, CA),
Upper strand: CACCGGCTGGAAACGCGAGATCCCGAAGGATCTCGCGTTTCCAGCC,
Lower strand: AAAAGGCTGGAAACGCGAGATCCTTCGGGATCTCGCGTTTCCAGCC
The upper and lower strands were annealed and sub-cloned into entry vector pENTR/H1/TO vector (Invitrogen, Carlsbad, CA) and recombinant lentivirus was produced using Virapower T-Rex Lentiviral expression system (Invitrogen, Carlsbad, CA) following manufacture’s instruction. 20 MOI were used to knock down the endogenous MLK3 in MEFs.
For immunoblotting, equal protein content of cell extracts or the immunoprecipitated protein samples were taken. The proteins were separated on denaturing SDS-PAGE and transferred onto polyvinylidene difluoride membrane (PVDF) and blotted with antibodies as indicated. Antibodies used were: anti-FLAG (Sigma, St. Louis, MO); anti-GST (Millipore, Billerica, MA); anti-TRAF2 (Cell Signaling, Danvers, MA); anti-JNK (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phospho JNK (Promega, Madison, WI). The antibody for immunoprecipitating endogenous JNK was provided by Dr. Joseph Avruch (Massachusetts General Hospital, Boston, MA). For immunoprecipitating endogenous MLK3, the antibody against the C-terminus peptide of MLK3 was developed in our laboratory, as described in [24, 33]. For blotting endogenous MLK3, the antibody against the N-terminal peptide of MLK3, developed in our laboratory was used.
Jurkat T cells, TRAF2+/+, TRAF2-/- MEFs and HEK293 cells were lysed in lysis buffer as described previously . The cell extracts were clarified by centrifugation at 15,000 × g for 5 min, and protein contents were estimated using the Bradford method. Immunoprecipitation and glutathione S-transferase (GST) pull-down assays were performed either by specific antibodies or glutathione-Sepharose beads, respectively. After thorough washing, the immunoprecipitates were either processed for immunoblotting or used for kinase assay as described previously [2, 5].
We thank Dr. James R. Woodgett for providing the TRAF constructs and TRAF2 knockout MEFs. This work was supported by National Institutes of Health (NIH) Grant GM55835 and Veterans Affairs Merit Award (to AR). BR is supported through NIH support (CA121221) and Veterans Affairs Merit Award.