NF-κB (nuclear factor κB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-κB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-κB has given another layer of complexity in NF-κB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-κB 2) as an inhibitor of NF-κB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKγ, a non-kinase component of the IκB (inhibitory κB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKγ and prevented the association of IKKγ with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKγ. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKγ and, consequently, failed to inhibit NF-κB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKγ. The RIP mutant with deletion of this similar region did not associate with IKKγ and had substantial reduction of its ability to mediate NF-κB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-κB signalling pathway through the interaction with IKKγ. Finally, the signalling pathway of NF-κB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-κB.
Abbreviations used: ABIN-2, A20 binding inhibitor of nuclear factor κB 2; CARD, caspase recruitment domain; CMB, core motif for binding; CYLD, cylindromatosis; DMEM, Dulbecco's modified Eagle's medium; FLIP1, foetal liver LKB1-interacting protein; GST, glutathione S-transferase; IκB, inhibitory κB; IKK, IκB kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NF-κB, nuclear factor κB; RIP, receptor-interacting protein; TNF, tumour necrosis factor; TNFR, TNF receptor; TRADD, TNFR1-associated death-domain protein.