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. 1999 Feb 16;96(4):1234-9.
doi: 10.1073/pnas.96.4.1234.

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

N Tsukamoto  1 , N Kobayashi, S Azuma, T Yamamoto, J Inoue
Affiliations

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

N Tsukamoto et al. Proc Natl Acad Sci U S A. .

Abstract

CD40 signaling modulates the immune response at least in part by activation of nuclear factor kappaB (NFkappaB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFkappaB. 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 NFkappaB 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 NFkappaB 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, NFkappaB activation by cyt-C is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFkappaB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFkappaB 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.

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Figures

Figure 1
Figure 1
TRAF2, TRAF3, and TRAF5 bind to the carboxyl-terminal conserved domain, but TRAF6 binds to the N-terminal domain in CD40cyt. (A) Alignment of human and mouse CD40cyt. The carboxyl-terminal part of CD40cyt is identical between hCD40 and mCD40, but the N-terminal part is poorly conserved. Conserved residues substituted in mutagenesis described in the text are indicated by boxes, and the resulting aas by the mutagenesis are shown above these boxes. GST-tagged hCD40cyt constructs (cyt, cyt-N and cyt-C) are shown below the sequences. (B) In vivo association of TRAFs with GST-hCD40 in 293T cells. Cell extracts from 293T cells cotransfected with expression plasmids for Flag-tagged TRAFs and GST-tagged hCD40cyt or its deletion mutants were prepared. The GST pulldown assays were performed and TRAF proteins bound to beads were then analyzed by Western blotting by using anti-Flag antibody M2 (Top). One-tenth of GST fusion proteins attached to beads was separated on 12.5% polyacrylamide/SDS gel and visualized by Coomassie brilliant blue R-250 staining (Bottom). A portion of the lysate used for the GST pulldown assay was separated on 8.5% polyacrylamide/SDS gel and expression level of Flag-tagged TRAFs was analyzed by Western blotting by using anti-Flag antibody M2 (Middle).
Figure 2
Figure 2
Glu-235 in hCD40 is critical for TRAF6 binding. (A) TRAF6 binding with cyt-N was abolished by the QE234–5AA mutation. Conserved residues in the N-terminal poorly conserved region in hCD40cyt were mutated in the cyt-N deletion mutant, and each construct was tagged with GST. GST-tagged CD40 cytoplasmic domain or its mutants and Flag-tagged TRAF6 were transiently expressed in 293T cells and in vivo binding assays were performed. Association between Flag-TRAF6 and GST-tagged CD40 mutants (Top) and expression level of Flag-TRAF6 (Middle) and that of GST-tagged CD40 mutants (Bottom) are shown. (B) Single point mutation E235A abolished TRAF6 association with GST-CD40. (Left) Either Gln-234 or Glu-235 alone was mutated to Ala in the GST-tagged cyt-N deletion mutant. GST-tagged CD40cyt or its mutants and Flag-tagged TRAF6 were transiently coexpressed in 293T cells and in vivo binding assays were performed. Association between Flag-TRAF6 and GST-tagged CD40 mutants (Top) and expression level of Flag-tagged TRAF6 (Middle) and that of GST-tagged CD40 mutants (Bottom) are shown. (Right) Glu-239 in mCD40, corresponding to Glu-235 in hCD40, was mutated to Ala in GST-tagged full-length cyt of mCD40. In vivo binding assay between mCD40 and TRAF6 in 293T cells was performed as described above.
Figure 3
Figure 3
Effects of E235A and T254A mutations on TRAF6 or TRAF2 association with GST-cyt. GST-cyt or its mutants (E235A or T254A) and Flag-tagged TRAF6 or TRAF2 were transiently expressed in 293T cells and binding assays were performed. Association of Flag-tagged TRAF6 and Flag-tagged TRAF2 with GST-tagged CD40 mutants (Top) and expression level of Flag-tagged TRAF6 and Flag-tagged TRAF2 (Middle) and that of GST-tagged CD40 mutants (Bottom) are shown.
Figure 4
Figure 4
Effects of E235A and T254A mutations on the CD40-mediated NFκB activation. (A) NFκB activation by CD40cyt-N was completely abolished by the E235A mutation. (Upper) CD40L-dependent fold activation of NFκB by CD40cyt-N (closed bar) and that by CD40cyt-N E235A (open bar) are shown. (Lower) CD40L-dependent fold activation of NFκB by CD40cyt-C (closed bar) and that by CD40cyt-C T254A (open bar) are shown. (B) Effects of E235A and T254A mutations on NFκB activation by the full-length CD40. CD40L-dependent fold activation of NFκB by the full-length CD40 with various mutatons is shown. Jurkat cells (2 × 106) were transfected with 0.5 μg of 3xκB-Luc, 0.5 μg of β-actin-β-gal, the indicated amounts of pME-hCD40 construct, 0 μg or 0.5 μg of pME-hCD40L, and enough pME18S control plasmid to give 5 μg of total DNA by the DEAE-dextran method. Cell extracts prepared at 36 hr after transfection were used for the luciferase assay. Fold activation was expressed as the ratio of luciferase activity with CD40L expression to that without CD40L expression. Values correspond to means ± SEMs of at least three independent experiments.
Figure 5
Figure 5
Effect of kinase-negative NIK on the NFκB activation by TRAF or CD40. 293T cells (4 × 105) were transfected with 1 ng of 3xκB-Luc, 10 ng of β-actin-β-gal, and various expression vectors by the calcium phosphate method. The total amount of DNA transfected was adjusted to 10 μg with a control expression vector. Thirty-six hours after transfection, cell extracts were prepared and used for luciferase assay. (A) Kinase-negative NIK inhibits NFκB activation by TRAF2 or TRAF5 more efficiently than that by TRAF6. pME-Flag TRAF2 (2 μg), TRAF5 (2 μg), or TRAF6 (1 μg) and indicated amounts of pME-NIK(KK429–430AA) were transfected. Luciferase activity in the absence of kinase-negative NIK was set to 100. Actual luciferase activities induced by each TRAF protein in the absence of kinase-negative NIK were comparable: TRAF2 (5210 ± 150), TRAF5 (4930 ± 110), TRAF6 (6650 ± 180). Expression levels of each TRAF protein in transfected cells were measured by Western blotting by using anti-Flag antibody (Inset). (B) Kinase-negative NIK inhibits NFκB activation by CD40cyt-C more efficiently than that by CD40cyt-N. 0.03 μg of pME-hCD40cyt-N or pME-hCD40cyt-C and 0 μg or 0.03 μg of pME-hCD40L were transfected. CD40L-dependent fold activation of NFκB is shown. Relative values in which fold activation in the absence of kinase-negative NIK was set to 100 are shown below each column. Values correspond to means ± SEMs of at least three independent experiments.
Figure 6
Figure 6
A model for NFκB activation by the CD40cyt. There are two domains (cyt-N and cyt-C) in the CD40cyt for the NFκB activation. The cyt-N domain activates the NFκB through TRAF6 and the contribution to the activation by this domain is approximately 70%, whereas the cyt-C domain activates the NFκB through TRAF2 and/or TRAF5 and the contribution to the activation is approximately 30%. Although the NFκB activation by the cyt-C domain is thought to be mediated mainly by NIK, it is possible that the NFκB activation by cyt-N is mediated by an unknown molecule (kinase) in addition to NIK.
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