A20 Inhibits Tumor Necrosis Factor (TNF) Alpha-Induced Apoptosis by Disrupting Recruitment of TRADD and RIP to the TNF Receptor 1 Complex in Jurkat T Cells

The regulation of survival and death in lymphoid cells is a tightly regulated process, and alteration of the delicate balance between the two can lead to a variety of autoimmune and inflammatory diseases. Members of the tumor necrosis factor (TNF) superfamily are critical regulators of the balance between the two opposing processes, and binding to their cognate receptors can trigger signaling pathways leading to either survival or death (25). Indeed, in the case of TNF receptor 1 (TNFR1/CD120a), ligation of a single class of receptor can trigger either cell survival or apoptosis depending on which signaling pathway predominates in the cell type examined. Studies conducted over the past several years have identified a number of molecules that are involved in the proximal signaling mechanism of TNFR1. Upon binding of TNF alpha to TNFR1, the death domain present in the cytoplasmic tail of the receptor is able to recruit and associate with two death domain-containing signaling molecules known as TRADD (TNFR1-associated death domain) and RIP (receptor-interacting protein) (13-15, 45). However, the mechanisms by which these death domain interactions are regulated remain poorly understood. Both TRADD and RIP are required for signaling to NF-κB, as well as the apoptosis pathway (13-15, 18, 36, 42). In turn, TRADD and RIP associate with TRAF2 and another death domain protein called FADD (FAS-associated death domain protein) (13, 14, 41, 45). It is at this juncture that the NF-κB and apoptotic signals are thought to bifurcate: TRAF2 recruitment leads to the activation of downstream kinases and NF-κB activation, whereas FADD recruitment leads to the activation of caspase 8 and apoptosis (6, 14, 24). In most instances, TNF binding to TNFR1 induces an NF-κB response but does not result in apoptosis unless the cells are simultaneously treated with protein synthesis inhibitors. The molecular basis for this observation was subsequently explained by the demonstration that cells lacking NF-κB transcription factors became sensitive to TNF-induced apoptosis and that this was due to the lack of expression of NF-κB-dependent antiapoptotic genes (2, 24, 43, 47). It appears that the NF-κB pathway triggered by TNFR1 antagonizes the apoptosis pathway that is simultaneously activated. However, the identities of the antiapoptotic genes that are regulated by NF-κB and the mechanisms by which these molecules inhibit TNF-mediated apoptosis remain to be fully explored. In this study, we address the above question by using a Jurkat T-cell mutant that we isolated that is defective in I-κB kinase γ (IKKγ)/NEMO expression. Inactive NF-κB transcription factors in the cytoplasm are bound to inhibitory I-κB proteins, and upon stimulation, the I-κB is phosphorylated on specific serine residues by IKK (17). This phosphorylation event targets I-κB for ubiquitination and degradation by the proteosome, thereby allowing the free NF-κB factors to translocate to the nucleus. IKK is a multimeric complex, and at its core, it consists of two catalytic subunits, IKKα and IKKβ, and an essential regulatory subunit, IKKγ/NEMO (10, 11, 26, 37, 38, 49-51). As a result of the IKKγ deficiency, the mutant Jurkat T-cell line that we isolated lacks a functional IKK and is unable to activate NF-κB (11, 50). In the absence of the protective effects of NF-κB, these IKKγ-deficient cells now became highly sensitive to TNF-induced apoptosis compared to their wild-type counterpart. We sought to identify the antiapoptotic genes that are regulated by NF-κB in our Jurkat T-cell model. Our analyses indicate that the zinc finger protein A20 is not induced in the IKKγ-deficient cells and that its ectopic expression in the mutant cells was sufficient to confer resistance to TNF-mediated cell death. Furthermore, we show here that A20 blocks the TNF apoptosis pathway at a very early step by inhibiting the recruitment of the death domain-containing signaling molecules TRADD and RIP to the receptor.