An 11-amino acid sequence in the cytoplasmic domain of CD40 is sufficient for activation of c-Jun N-terminal kinase, activation of MAPKAP kinase-2, phosphorylation of I kappa B alpha, and protection of WEHI-231 cells from anti-IgM-induced growth arrest

We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35-45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of I kappa B alpha phosphorylation and degradation. Amino acids 35-45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-kappa B, JNK, and p38 MAPK.     

The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.     

1Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.     

Tumor necrosis factor (TNF) receptor-associated factor 7 is required for TNFα-induced Jun NH2-terminal kinase activation and promotes cell death by regulating polyubiquitination and lysosomal degradation of c-FLIP protein

The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.     

TNF-receptor I defective in internalization allows for cell death through activation of neutral sphingomyelinase

The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI DeltaTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI DeltaTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI DeltaTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis.     

NESK, a member of the germinal center kinase family that activates the c-Jun N-terminal kinase pathway and is expressed during the late stages of embryogenesis

The c-Jun N-terminal kinase (JNK) signaling pathway plays a crucial role in cellular responses stimulated by stress-inducing agents and proinflammatory cytokines. The group I germinal center kinase family members selectively activate the JNK pathway. In this study, we have isolated a mouse cDNA encoding a protein kinase homologous to Nck-interacting kinase (NIK), a member of the group I germinal center kinase family. This protein kinase is expressed during the late stages of embryogenesis, but not in adult tissues, and thus named NESK (NIK-like embryo-specific kinase). NESK selectively activated the JNK pathway when overexpressed in HEK 293 cells but did not stimulate the p38 kinase or extracellular signal-regulated kinase (ERK) pathways. NESK-induced JNK activation was inhibited by the dominant negative mutants of MEKK1 and MKK4. Tumor necrosis factor (TNF)-alpha or TNF receptor-associated factor 2 (TRAF2) stimulated the NESK activity. Furthermore, the dominant negative NESK mutant inhibited the JNK activation induced by TNF-alpha or TRAF2. These results suggest that NESK, a novel activator of the JNK pathway, functions in coupling TRAF2 to the MEKK1 --> MKK4 --> JNK kinase cascade during the late stages of mammalian embryogenesis.     

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-alpha signaling.

Tumor necrosis factor-alpha (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-kappa B, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-kappa B but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-kappa B and antiapoptosis.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaB-inducing kinase. Identification of a novel TRAF6 interaction motif

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappaB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565-568 and 606-611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappaB activation. However, RANK-mediated JNK activation required a distal portion (427-603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.     

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.     

Tumor necrosis factor receptor 2 (TNFR2) signaling is negatively regulated by a novel, carboxyl-terminal TNFR-associated factor 2 (TRAF2)-binding site

Tumor necrosis factor (TNF) superfamily receptors typically induce both NF-kappaB and JNK activation by recruiting the TRAF2 signal transduction protein to their cytoplasmic domain. The type 2 TNF receptor (TNFR2), however, is a poor activator of these signaling pathways despite its high TRAF2 binding capability. This apparent paradox is resolved here by the demonstration that TNFR2 carries a novel carboxyl-terminal TRAF2-binding site (T2bs-C) that prevents the delivery of activation signals from its conventional TRAF2-binding site (T2bs-N). T2bs-C does not conform to canonical TRAF2 binding motifs and appears to bind TRAF2 indirectly via an as yet unidentified intermediary. Specific inactivation of T2bs-N by site-directed mutagenesis eliminated most of the TRAF2 recruited to the TNFR2 cytoplasmic domain but had no effect on ligand-dependent activation of the NF-kappaB or JNK pathways. By contrast, inactivation of T2bs-C had little effect on the amount of TRAF2 recruited but greatly enhanced ligand-dependent NF-kappaB and JNK activation. In wild-type TNFR2 therefore, T2bs-C acts in a dominant fashion to attenuate signaling by the intrinsically more active T2bs-N but not by preventing TRAF2 recruitment. This unique uncoupling of TRAF2 recruitment and signaling at T2bs-N may be important in the modulation by TNFR2 of signaling through coexpressed TNFR1.     

Membrane localization of TRAF 3 enables JNK activation

Members of the tumor necrosis factor receptor family as well as other receptors achieve their diverse biological effects through the activation of intracellular signals including the c-Jun N-terminal kinase (JNK) pathway. Such signals are believed to be delivered through mediators known as TNF receptor-associated factors (TRAFs). Although the N-terminal zinc finger region of TRAFs has been shown to be essential for downstream signaling, there is no indication yet as to the nature of its role or of the factors that distinguish the N terminus of TRAF 3, which does not activate JNK in the systems examined thus far, from those of other TRAFs, which do activate this pathway. In the present study, it is shown that, among the known TRAFs, localization to the insoluble cell pellet fraction consistently correlates with JNK activation and that both characteristics map to the TRAF N terminus. Furthermore, it is demonstrated that forced localization of TRAF 3 to the cell membrane is sufficient to convert this molecule into an activator of JNK. This suggests that one of the roles of the TRAF N terminus may be to participate in interactions that promote the recruitment of TRAFs to the membrane and that this localization effect plays an important role in TRAF-mediated JNK activation.     

Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase

We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.