TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation:: implications for interaction with NEMO/IKKγ and NF-κB activation

Pro-inflammatory cytokines trigger signalling cascades leading to NF-kappa B (nuclear factor-kappa B)-dependent gene expression through IKK [I kappa B (inhibitory kappa B) kinase]-dependent phosphorylation and subsequent degradation of the I kappa 3 proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNF alpha (tumour necrosis factor alpha) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-kappa B activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKK beta upon TNF alpha stimulation and that this modification negatively regulates TANK binding to NEMO (NF-kappa B essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNF alpha-mediated induction of a subset of NF-kappa B target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNF alpha by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKK beta-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-kappa B activation.