Molecular determinants of NF-kappaB-inducing kinase action.
NF-kappaB 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 IkappaB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB 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-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaB alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB 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 IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. 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 IKKalpha; 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-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. 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.
Lin
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X
Gladstone Institute of Virology and Immunology, Microbiology and Immunology, University of California, San Francisco, California 94141, USA.
Mu
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Cunningham
E T
ET
Marcu
K B
KB
Geleziunas
R
R
Greene
W C
WC
D000595:Amino Acid Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000097782:NF-kappaB-Inducing Kinase
D051550:I-kappa B Kinase; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D013912:Threonine; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human