Two differently regulated nuclear factor κB activation pathways triggered by the cytoplasmic tail of CD40

CD40 signaling modulates the immune response at least in part by activation of nuclear factor KB (NFκB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFκB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFκB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFκB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFκB activation by cyt-C is inhibited by a kinase-negative form of NFκB- inducing kinase more efficiently than that by cyt-N, consistent with the result that NFκB activation by TRAF2 and TRAF5 is inhibited by a kinase- negative form of NFκB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFκB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.