Tumor Necrosis Factor (TNF) Receptor 1 Signaling Downstream of TNF Receptor-associated Factor 2

Like other members of the tumor necrosis factor (TNF) receptor family, p55 TNF receptor 1 (TNF-R1) lacks intrinsic signaling capacity and transduces signals by recruiting associating molecules. The TNF-R1 associated death domain protein interacts with the p55 TNF-R1 cytoplasmic domain and recruits the Fas-associated death domain protein (which directly activates the apoptotic proteases), the protein kinase receptor interacting protein, and TNF receptor-associated factor 2 (TRAF2). TRAF2 has previously been demonstrated to activate both transcription factor nuclear factor κB (NFκB) and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, which in turn stimulates transcription factor activating protein 1 (AP1) mainly via phosphorylation of the c-Jun component. We have investigated the signaling properties of NFκB-inducing kinase (NIK), a TRAF2-associated protein kinase that mediates NFκB induction. NIK was found to be unable to activate JNK/SAPK, mitogen-activated protein kinase, or p38 kinase. Moreover, NIK was not required for JNK/SAPK activation by TNF-R1, thus representing the first TNF-R1 complex component to dissect the NFκB and the JNK/SAPK pathways. Despite being unable to activate JNK/SAPK and mitogen-activated protein kinase, NIK strongly activated AP1 and was required for TNF-R1-induced AP1 activation. Therefore, NIK links TNF-R1 to a novel, JNK/SAPK-independent, AP1 activation pathway.

Lysates were cleared by centrifugation, and protein concentration was measured using a commercial Bradford protein assay (Promega). Equal amounts of each lysate (usually 500 g) were incubated on ice with 2 g of anti-HA antibody 12CA5 (Boehringer) (JNK assays) or anti-FLAG antibody (IBI Kodak) (p38 assays) for 2 h. Immune complexes were collected by protein A-agarose for 25 min, washed thrice with radioimmune precipitation buffer containing 20 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 0.5 mM DTT, and washed once with kinase reaction buffer (20 mM Hepes, pH 7.5, 20 mM MgCl 2 , 20 mM ␤-glycerophosphate, 2 mM DTT, 100 M sodium orthovanadate, 0.5 mM sodium fluoride). Samples were finally resuspended with 40 l of kinase reaction buffer containing 20 M ATP, 2.5 Ci of [␥-32 P]ATP and either 2 g of glutathione S-transferase-c-Jun (1-141) (JNK assays) or 8 g of mielin basic protein (p38 assays) and incubated at 30°C for 20 min. Reactions were stopped by the addition of 3 ϫ Laemmli sample buffer; samples were boiled and loaded on 12.5% SDS-acrylamide gels. After fixing and drying, gels were autoradiographed at Ϫ70°C. Radioactivity in each spot was quantitated with a PhosphorImager. The amount of exogenous transfected kinase in each sample was analyzed by Western blotting. Reporter gene assays were performed as described (24).

RESULTS AND DISCUSSION
In both 293 and HeLa cells, TRADD, TRAF2, and NIK are able to induce a strong NFB activation (NIK being the strongest activator) (Fig. 1, A and B). Deletion of the N-terminal (putatively regulatory) domain of NIK (NIK⌬1234) does not apparently affect NFB activation, whereas the deletion of the catalytic domain (NIK⌬2101) or its inactivation (Lys 3 Arg mutation at aa 429) abolish NFB activation (Fig. 1B). Consistent with a requirement for NIK in TNF-induced NFB activation, overexpression of a C-terminal NIK fragment (NIK⌬2101), which binds TRAF2 and presumably blocks the recruitment of endogenous NIK and/or titrates downstream effectors (23), significantly impairs the induction of NFB by either TNF treatment or overexpression of TNF-R1 complex components (Fig. 1C). These data indicate that NIK is required for the activation of NFB by TNF-R1/TRAF2 in different cell types.
Because TRAF2 overexpression is sufficient to activate both NFB and JNK/SAPK (13-16), we examined whether NIK is able to activate JNK/SAPK as well. NIK was cotransfected in HeLa and 293 cells together with a hemagglutinin (HA)-tagged SAPK␥ expression vector, and the activity of exogenous transfected SAPK was assayed 36 -48 h after transfection. In 293 cells, wherein TRAF2 usually gives the highest activation of SAPK/JNK, neither NIK or NIK⌬1234 (which are both efficient NFB activators) were able to elevate JNK/SAPK activity over the baseline. In a similar manner, we were unable to detect any JNK/SAPK activation by NIK in HeLa cells (Fig. 2, A and B). Similarly to JNK/SAPK, p38 activation by TNF depends on TRAF2 (14) 2 but is not dependent on NIK (Fig. 2C).
Apart from inducing a prolonged activation of JNK/SAPK and p38, TNF-R1 engagement provokes a mild and transient activation of the mitogen-activated protein kinase (MAPK), whose biological role has not been defined (26,27). MAPK activation by TNF may depend on a TNF-R1 domain that is distinct from the TRADD interaction domain and that interacts with a recently identified protein known as FAN (28). Consistent with MAPK activation being a TRADD-independent function, neither TRAF2 or NIK were able to activate MAPK in the cells tested (Fig. 2D).
The effects of the dominant negative NIK mutant (NIK⌬2101) on JNK/SAPK activation by TNF-R1 were next evaluated. The expression of ⌬2101 at levels that gave maximal inhibition of NFB induction (Fig. 1C) did not impair the ability of either TNF or TRAF2 to activate JNK/SAPK (Fig. 2E). Therefore, when the NIK pathway is blocked by expression of dominant negative NIK, both receptor cross-linking and overexpressed TRAF2 still activate JNK/SAPK. Taken together our results suggest that: (i) NIK is neither sufficient nor required for JNK/SAPK activation by TNF-R1/TRAF2; (ii) the bifurcation between the NFB pathway and the JNK/SAPK pathway occurs immediately downstream of TRAF2; and (iii) dominant negative NIK does not disrupt the TNF-R1 complex nonspecifically. Therefore, NIK dissects the TRAF2 pathway leading to NFB activation from the pathway leading to JNK/SAPK activation; this suggests that the  (32). C, dominant negative NIK blocks AP1 activation by TNF-R1. HeLa cells were transfected with Ϫ73/ϩ63 ColCAT together with NIK⌬2101. Expression of NIK⌬2101 severely reduced AP1 activation by both TNF and overexpressed TRAF2 without any significant effect on MEKK-induced AP1 activity.

FIG. 2. JNK/SAPK activation by TNF-R1 is independent of NIK.
The effects of NIK expression on JNK/SAPK activity were evaluated in HeLa (A) and 293 cells (B). HA-p46SAPK␥-pcDNA3 was cotransfected in both cell lines together with the NIK expression vectors described above. 48 h after transfection, HA-SAPK␥ was immunoprecipitated with a monoclonal anti-HA antibody (12CA5), and its activity was determined using glutathione S-transferase-Jun as substrate. JNK/ SAPK activation by TRAF2 and the effect of dominant negative TRAF2 on JNK/SAPK activation by hrTNF␣ (1000 IU/ml, 15 min) are also shown. A sample of each lysate was analyzed for expression of HA-SAPK␥ by Western blotting. Similar results were obtained when expression vectors for untagged or FLAG epitope-tagged NIK were used. C, p38 activity is not up-regulated by NIK in HeLa cells. HeLa cells were transfected with a FLAG-tagged p38 expression vector together with the NIK expression vectors described above. The activity of immunoprecipitated p38 was analyzed in a kinase assay using myelin basic protein (MBP) as substrate (18). D, overexpressed NIK does not activate the MAPK. HeLa cells were transfected with TRAF2 or NIK expression vector and serum starved 24 h after transfection. After additional 24 h, p42/44 MAPK/Erk activation was analyzed by immunoblotting using a rabbit polyclonal phospho-specific antibody (New England Biolabs) that recognizes p42 and p44 MAPK only when catalytically activated by phosphorylation at a critical Tyr residue. A constitutively activated Raf that is deleted of the N-terminal regulatory domain (Raf(BXB)) was used as a positive control. To show equal loading in all wells the same filter was rehybridized with a rabbit polyclonal anti-MAPK antibody (Upstate Biotechnology Inc.). E, blockade of the NIK pathway does not affect JNK/SAPK activation by TNF-R1/TRAF2. The effects of a dominant negative NIK mutant, NIK⌬2101, on SAPK␥ activation by TRAF2 or TNF were studied in HeLa cells. 48 h after transfection, cells were either left unstimulated or treated with hrTNF␣ (1000 IU/ml) for 15 min. Detergent lysates were prepared and processed as described above.
ability of TNF-R1/TRAF2 to activate JNK/SAPK must depend on a different TRAF2-interacting protein. One possible candidate is represented by MEKK1, a kinase that phosphorylates and activates SEK/JNKK, which in turn phosphorylates JNK/SAPK (29 -32). However, we have been unable to detect a physical interaction between TRAF2 and MEKK1. 2 Therefore, the evidence for a role of MEKK1 in TNF-R1 signaling is indirect and arises from the ability of catalytically inactive MEKK1 (MEKK1-KM) to block TNF-R1/TRAF2-induced activation of SAPK/JNK (14); at this point we cannot exclude the possibility that SAPK/JNK activation by TNF-R1/TRAF2 depends on a putative MEKK1related protein whose activity is inhibited by MEKK1-KM overexpression.
The prolonged activation of JNK/SAPK by TNF and the consequent phosphorylation and activation of the c-Jun transcriptional activation domain correlate with the sustained induction of AP1-dependent genes (33,34). AP1 is composed of proteins of the Jun and Fos families that associate to form a variety of homo-and heterodimers that bind to a common recognition element known as either the tetradecanoic phorbol acetate-response element or the AP1 binding site (35); the presence of AP1 sites in the promoters of several genes, including those encoding for cytokines and adhesion molecules, contributes to the induction of such genes by TNF as well as by other AP1 inducers. With respect to TNF, TRAF2 (which is an efficient JNK/SAPK activator) was found to be a stronger stimulator of AP1-dependent transcription (Fig. 3A). Unexpectedly, overexpression of NIK, which activates neither JNK/SAPK nor MAPK, strongly activated transcription directed by a canonical AP1 site. This effect of NIK was dependent on an intact protein kinase domain. Moreover, AP1 activation by NIK was not blocked by dominant negative SEK/JNKK (Fig. 3B) or by chemical inhibitors of p38 and MAPK/Erk. 2 Evidence that NIK contributes to the induction of AP1 activity by TNF-R1/TRAF2 comes from experiments with dominant negative NIK; indeed, NIK⌬2101 was able to reduce TNF-induced activation of AP1 by more than 50% without any evident effect on basal AP1 activity (Fig. 3C). Therefore, when the NIK pathway is blocked and the JNK/SAPK pathway fully active (Fig. 2), the ability of TNF-R1 to stimulate AP1 activity is severely impaired. The effect of NIK⌬2101 on TRAF2-dependent activation of AP1 was slightly less evident; this may reflect a major contribution of the JNK/SAPK pathway to AP1 activation by overexpressed TRAF2, consistent with the greater potency of transfected TRAF2 compared with TNF in JNK/SAPK induction (Fig. 2).
TRAF2 is a critical signaling molecule that links both p55 TNF-R1 and p75 TNF-R2 to NFB and JNK/SAPK activation pathways. TRAF2-dependent activation of NFB is required for the induction of several genes, including those protecting cells from TNF-induced apoptosis (14,15,(37)(38)(39); conversely, the activation of JNK/SAPK does not seem to be relevant for cytotoxicity (14,15) but collaborates to the induction of adhesion molecules and cytokines (40). 2 The results reported here indicate that the NFB and the JNK/SAPK pathways bifurcate immediately downstream of TRAF2; this would suggest that TRAF2 signals by interacting with and activating at least two distinct effectors, namely NIK, which seems to be responsible for NFB activation (possibly through direct activation of the recently identified IB kinase) (41,42), and yet unknown transducers are responsible for JNK/SAPK activation. Apart from inducing NFB, NIK couples TNF-R1/TRAF2 to AP1 activating pathways that are alternative to MAPK and JNK/SAPK. The inhibitory effect of dominant negative NIK on AP1 activation by TNF suggests that this AP1 activation pathway is not redundant but probably collaborates with the JNK/SAPK and the MAPK pathways to achieve an optimal AP1 activation. The mechanisms of AP1 activation by NIK is still unclear. Because AP1 is a collection of dimers composed by several Fos and Jun family proteins, it is highly likely that a number of regulatory mechanisms other than c-Jun phosphorylation contribute to control its activity at various levels. The identification of downstream target(s) of NIK will help elucidate the mechanism of AP1 activation through this pathway.