Molecular determinants of NF-κB-inducing kinase action

NF-kappa B corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed I kappa B, Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappa B expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappa B-inducing kinase (NIK) and I kappa B-specific kinases (IKK alpha and IKK beta), When activated, IKK alpha and IKK beta directly phosphorylate the two N-terminal regulatory serines within I kappa B alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome, This process liberates the NF-kappa B complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKK alpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappa B. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKK alpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947, When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappa B and alone is sufficient to disrupt the physical association of NIK and IKK alpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.