Previous studies have demonstrated that the TAK1-associating protein TAB2 interacts with TRAF6 in an IL-1-dependent manner, resulting in the formation of a TRAF6-TAB2-TAK1 complex (36
). Formation of this complex appears to be required for IL-1-mediated activation of JNK and NF-κB. In fact, mutational analysis has revealed that the integrity of the TAB2 protein is essential not only for activating downstream signals but also for mediating the association of TAK1 with TRAF6. In addition, our previous results have shown that once IL-1 signaling is initiated, the membrane pool of TAB2 translocates to the cytosol, where it mediates the interaction between TRAF6 and TAK1 (36
). Therefore, in our present picture, TAB2 acts as an adapter that links TAK1 and TRAF6 and thereby mediates the activation of TAK1 in the IL-1 signaling pathway. By this model, the redistribution of TAB2 proteins upon IL-1 stimulation is a key step in the specific activation of TAK1. In addition, IRAK is essential for the activation of NF-κB and JNK by IL-1 and functions upstream of TRAF6 in the IL-1 pathway (13
). However, the role of IRAK in this signal transduction cascade has previously not been defined. In this study, we examined how IRAK mediates the activation of TAK1 in response to the IL-1 signal.
Several lines of evidence lead us to propose that IRAK plays a critical role in the formation of the TRAF6-TAB2-TAK1 complex by inducing the translocation of TAB2. First, IL-1 treatment induces the association of IRAK-TAB2, IRAK-TRAF6, TRAF6-TAB2, and TRAF6-TAK1, each with similar kinetics, consistent with the idea that IL-1 induces the formation of a multicomponent complex. Second, in IRAK-deficient cells, TAB2 translocation to the cytosol and its association with TRAF6 in response to IL-1 are abolished. These results indicate that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of the TRAF6-TAB2 complex. Third, TAK1 activation occurs rapidly following IL-1 application. The kinetics of TAK1 activation following IL-1 stimulation parallels the observed formation of complexes among IRAK, TRAF6, TAB2, and TAK1. It therefore seems reasonable to assume that formation of the TRAF6-TAB2-TAK1 complex constitutes an early event in the activation of TAK1 by IL-1. Finally, IL-1 stimulation does not induce activation of TAK1 in IRAK-deficient cells, demonstrating that IRAK indeed is essential for TAK1 activation in response to IL-1. Taken together, these results suggest a model in which IRAK functions in IL-1 signaling to facilitate the formation of the TRAF6-TAB2 complex. This model implies that IRAK-mediated relocalization of TAB2 plays an important role in IL-1 signaling. It remains to be determined, however, whether a large complex containing IRAK, TRAF6, and TAB2 is formed.
The innate immune mechanisms of host defense responses in vertebrates and Drosophila melanogaster
utilize remarkably conserved molecular components (11
). Analogous to IL-1 signaling, the Drosophila
Toll pathway leads to the phosphorylation and degradation of the IκB-like molecule Cactus, releasing the NF-κB-like transcription factor Dorsal to enter the nucleus. Dorsal activation signaled by Toll requires two intermediate signal transducers, Tube and Pelle (21
). Pelle is a serine/threonine kinase that is homologous to IRAK. Tube is an adapter molecule that tethers Pelle to the membrane (7
). Tube is therefore a functional homolog of MyD88, although these two molecules are not structurally related. A Drosophila
homolog of TRAF6 was recently identified (24
), leading to speculation that TRAF may also be involved in the Drosophila
Toll pathway. Furthermore, a novel evolutionarily conserved protein, Pellino, associates with Pelle and is thought to link between Pelle and the downstream target (10
). This suggests that Pellino and TAB2 may carry out analogous functions.
It has been reported that IRAK serine/threonine kinase activity is not essential for IL-1 signaling (16
). If so, what then might be the role of IRAK kinase activity in IL-1 signaling? Although cells lacking IRAK are defective in IL-1-induced activation of TAK1, the defect can be reversed by expression of either wild-type or catalytically inactive IRAK. It has been therefore suggested that the primary function of IRAK is to provide a scaffolding function and to facilitate the formation of signaling complexes during IL-1-mediated signaling. One idea is that IRAK kinase activity plays a role in IL-1 signal termination, rather than transduction. It is known that IRAK autophosphorylation is followed by proteolytic degradation (46
). Upon initial IL-1 stimulation, IRAK forms an immunocomplex with TAB2 and TRAF6 within 2 to 5 min, but these interactions disappear within 20 min after treatment. Since there is no significant change in IRAK protein levels during the first 20 min after IL-1 treatment, degradation of IRAK cannot account for the loss of these associations. Following IL-1 stimulation, TAB2 mobility in SDS-PAGE gels is altered. Phosphatase treatment indicated that this mobility shift is due to phosphorylation (36
). Although the biological significance is unclear at present, we speculate that phosphorylation of TAB2 may weaken its affinity for the TRAF6-TAB2 complex, thereby promoting dissociation of the complex. If this assumption is correct, it would suggest that TAB2 is modified by one of the kinases in the signal transduction pathway, such as IRAK. It will therefore be important to study the function of phosphorylated TAB2, to identify which amino acids are phosphorylated, and to investigate how phosphorylation is regulated.
IL-1-induced activation of endogenous TAK1 is transient, with activation occurring soon after IL-1 stimulation and terminating 15 min later. TAK1 is known to be activated by autophosphorylation in response to IL-1 (15
); however, TAK1 phosphorylation persists even after its kinase activity has diminished. This suggests that some other kinase(s) may phosphorylate TAK1. Indeed, we have previously observed that IKKs also phosphorylate TAK1 (30
). Based on these observations, one possible mechanism by which TAK1 is downregulated following its activation by IL-1 is the activation of IKKs, which in turn phosphorylate and inactivate TAK1. Consistent with this hypothesis, IKKα is still active after TAK1 activation terminates.
The existence of specific anchors or scaffolds that localize the components of signaling pathways may confer selectivity on kinase action, providing a means by which the same kinase can be involved in different signaling cascades. The action of protein kinases can be modulated by protein-protein interactions. In the case of the IL-1 signaling pathway, TAK1 binds selectively to TAB2, which functions as an adapter to link TAK1 to the TRAF6 complex. TRAF6 has further been shown to bind at least two other proteins involved in the IL-1 pathway: ECSIT, identified as a TRAF6-binding protein in a two-hybrid screen (17
), and p62, a protein that interacts with atypical protein kinase C (aPKC) (33
). ECSIT also interacts with MEKK1, which has been implicated in the activation of NF-κB through the phosphorylation of the IKK complex (19
). The aPKCs have been implicated in the activation of the IKK complex (18
). Therefore, there is a remarkable functional similarity among TAB2, ECSIT, and p62; each of these proteins binds TRAF6 and interacts with a kinase proposed to act upstream of the IKK complex, i.e., TAK1, MEKK1, and aPKC, respectively. These adapter proteins may therefore function to maintain specific regulation and suppress cross talk among signaling pathways.