Differential Requirements for Tumor Necrosis Factor Receptor-associated Factor Family Proteins in CD40-mediated Induction of NF-κB and Jun N-terminal Kinase Activation

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF- Kappa B and Jun N-terminal kinase (JNK) implying that CD40 can stimulate TRAF2- and TRAF3- independent pathways for NF- Kappa B and JNK activation. A carboxyl- truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding CD40( Delta 32), mediated NF- Kappa B induction through a mechanism that was suppressible by co-expression of TRAF6( Delta N), a dominant-negative version of TRAF6, but not by TRAF2( Delta N), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6( Delta N) did not impair JNK activation by CD40( Delta 32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF- Kappa B induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.