We previously reported that point mutations in the amino-terminal zinc ring finger domain of TRAF5 abolished its ability to mediate NF-κB activation but did not affect its ability to activate SAPK. In addition, we found that TRAF2A, an alternative spliced form of TRAF2 with a 7-aa insertion in the zinc ring finger domain of TRAF2, activates the SAPK pathway but not the NF-κB pathway (10
). We therefore suggested that TRAF proteins are the last common molecules between the NF-κB and SAPK pathways and may branch these two distinct pathways through different TRAF complexes. TRAF proteins have been reported to associate with many intracellular proteins, including TRADD, RIP, IRAK, NIK, TANK, TRIP, Peg3/Pw1, c-IAP1, c-IAP2, and A20 (4
). These proteins potentially form large TRAF-associated complexes important in TRAF-mediated cell signaling. Nevertheless, the roles and the mechanisms of these molecules in TRAF-mediated NF-κB and SAPK activation are not known.
The ability of TANK to modulate TRAF2-mediated NF-κB activation suggested that TANK could potentially function as either an activator or an inhibitor of NF-κB activation induced by TNFR family proteins. In this report, we showed that TANK synergistically activated SAPK with TRAF proteins. TANK-mediated synergistic SAPK activation varies with individual TRAF proteins. It strongly enhanced TRAF2-mediated SAPK activation in a dose-dependent manner but did not synergize with TRAF3 in SAPK activation. As with TRAF2-mediated NF-κB activation, the effects of TANK upon TRAF5- and TRAF6-mediated SAPK activation were biphasic, with enhancement by lower levels of TANK and decreased enhancement by higher levels. These results suggest that TANK might function as a regulatory molecule controlling the threshold of TRAF-mediated NF-κB and SAPK activation.
This report also describes a potential mechanism of TANK function during TRAF-mediated NF-κB and SAPK activation. We previously defined a 21-aa peptide in the middle of TANK as the TRAF family member-interacting motif in TANK (TIMtk), and showed that the TIMtk can compete with the TRAF binding motif in the CD40 cytoplasmic tail for binding to the TRAF-C domain of TRAF proteins (5
). In the work reported here, we predict that TANK forms a homodimer through an interaction in the TANK-N region, as well as forming an intramolecular association through the interaction between TANK-N and TANK-C, which may inhibit TANK dimerization. All these studies are consistent with our previously proposed model, suggesting that TANK in its ground state is autoinhibited by an intramolecular interaction of its carboxyl and amino termini. The binding of TRAF2 to the TIMtk motif in the middle of TANK may change TANK’s conformation, favoring the intermolecular TANK-N and TANK-N interaction to form a TANK dimer. Because TRAF binds TANK through the TIMtk, dimerization of TANK would lead to aggregation of TRAF2, which may trigger a signal for NF-κB and SAPK activation. Consistent with this model, we found that overexpression of TANK-C strongly inhibited NF-κB and SAPK activation induced by CD40, TRAF2 or TRAF2 plus TANK, presumably by competing away TANK dimerization sites at the amino terminus of endogenous TANK. Conversely, TANK-N overexpression synergistically activated TRAF-mediated NF-κB and SAPK activation, possibly by titrating away the autoinhibitory carboxyl-terminal portion of endogenous TANK.
Multiple pathways may be involved in SAPK activation. The SAPK activation initiated from many stress stimuli requires activation of a signaling cascade from the Rho group of small G proteins (such as Rac1 or cdc42) to mixed lineage kinases (such as MLK-2 and MLK-3), to MEKK1, to SEK1 or SEK2, to JNK family proteins, to c-Jun (3
). Other stress inducers may activate JNK through a family of germinal center kinases (25
). Recent studies suggested two potential signal transduction pathways for TRAF-induced SAPK activation (35
). One is mediated by ASK1 (or MAPKKK5), which is a serine/threonine kinase at the level of the MAPK kinase kinases (35
). Studies showed that ASK1 coprecipitates with TRAF2 upon TNF-α stimulation. The dominant-negative ASK1, which contains a lysine-to-alanine mutation at the ATP binding site, however, only partially inhibits TNF-α- or TRAF-induced SAPK activation (8
). Other studies suggest the involvement of GCK and GCKR in TRAF-induced SAPK activation (50
). In this report, we showed that although TRAF2 or TANK alone bound to GCKR weakly, together they formed a strong complex with GCKR. This synergistic binding of TRAF2 and TANK to GCKR may result in synergistic activation of SAPK by TRAF2 and TANK. Consistent with the role of GCKR in TRAF2- and TANK-mediated SAPK activation, we showed that the dominant-negative GCKR but not the dominant-negative Rac1 or cdc42 strongly inhibited SAPK activation induced by TRAF2 plus TANK. The GCK family proteins are kinases at the MAP4K level. Although their physiological targeting molecules in the kinase cascade are not yet confirmed, activation of some GCK family proteins is likely to lead to activation of MEKK1. We showed that the dominant-negative MEKK1 strongly inhibited SAPK activation induced by GCKR or GLK, whereas the dominant-negative GCKR or GLK did not affect MEKK-1-induced SAPK activation. Furthermore, coprecipitation between GCK and MEKK1 was recently reported (59
). The dominant-negative SEK1 abolished SAPK activation induced by upstream activators such as CD40, TRAF2, and TRAF2 plus TANK, suggesting that SEK1 may be a potential kinase involved in the TRAF-mediated SAPK activation pathway (8
). In addition to SAPK activation, TRAF proteins have also been reported to activate the p38 pathway, a stress-activated pathway parallel to the SAPK pathway (2
). Further studies are necessary to determine the contributions of ASK1 and GCK family proteins in TRAF- and TANK-mediated SAPK and p38 activation.
We also reported the endogenous activation of GCKR by CD40 in primary B cells. Dominant-negative TRAF2 and GCKR completely abrogated CD40-mediated SAPK activation in vivo. Together, these data suggest that GCKR is a critical mediator in CD40 activation of SAPK. Interestingly, the kinase known as GCK derived its name by virtue of its high expression in the follicular germinal center, with putative roles in B-cell differentiation and selection (22
). In B cells, CD40 has been implicated in a variety of functions, including germinal center formation, immunoglobulin isotype switching, antibody affinity maturation, and generation and maintenance of memory B cells (14
). However, how GCKR-mediated SAPK activation contributes to CD40-mediated B-cell functions is a critical and open question which should be addressed further. In addition to GCKR, our and other studies suggest that GLK, another GCK family protein, and GCK may also be involved in TNFR-mediated and in TRAF- and TANK-mediated SAPK activation. With 11 known members of the GCK family, it will be of interest to determine whether additional members are involved in signal transduction mediated by CD40 as well as other TNFRs. Specificity of GCK members to distinct TNFRs in vivo will be important in delineating the function of GCK family-mediated signaling.