Abstract
Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of kappaB kinase (IKK), as well as in inhibiting apoptosis induced by TNFalpha. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFalpha-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-DeltaR) has been widely used as a dominant negative in transient overexpression systems to block TNFalpha-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-DeltaR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFalpha-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells efficiently inhibited the TNFalpha-induced later phase of prolonged JNK activation, yet failed to inhibit TNFalpha-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-DeltaR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFalpha-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFalpha and is essential for suppressing the noncanonical nuclear factor kappaB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFalpha-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFalpha-induced cell death.