Methods to assess the activation of the alternative (noncanonical) NF-κB pathway by non-death TNF receptors.
The alternative or noncanonical NF-κB pathway regulates the generation of p52-containing NF-κB dimers (e.g., p52/RelB) through a partial degradation (called processing) of the precursor p100 into p52. This pathway is activated by a subset of non-death TNF receptor members, which ultimately activate two kinases: NIK (NF-κB-Inducing Kinase) and IKKα (Inhibitor of κB Kinase alpha). These kinases create a phosphodegron for the E3 ligase SCF-β-TrCP that covalently binds K48-linked polyubiquitin chain onto p100 prior to its proteasomal processing. The resulting p52-containing complexes translocate into the nucleus to activate target genes involved in secondary lymphoid organ development, B cell survival or in osteoclastogenesis. We describe in this chapter straightforward methods to monitor the activation of the alternative NF-κB pathway. These methods uncover cytosolic and nuclear biochemical modifications of key proteins of the alternative NF-κB pathway required prior to the transcription of NF-κB target genes.
Laboratory of Molecular Immunology and Signal Transduction, GIGA-Research, University of Liège, Avenue de l'Hôpital, 1, Sart-Tilman CHU, B34, 4000, Liège, Belgium.
Caroline
C
Remouchamps
Emmanuel
E
Dejardin
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D008024:Ligands; D016328:NF-kappa B / Q000378:metabolism*; D052003:NF-kappa B p52 Subunit / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D018124:Receptors, Tumor Necrosis Factor / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D048008:TNF Receptor-Associated Factor 3 / Q000378:metabolism; D051841:Transcription Factor RelB / Q000378:metabolism; D000097782:NF-kappaB-Inducing Kinase
D000911:Antibodies, Monoclonal; D051550:I-kappa B Kinase; D051034:Inhibitor of Apoptosis Proteins; D008024:Ligands; D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit; D017346:Protein Serine-Threonine Kinases; D018124:Receptors, Tumor Necrosis Factor; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3; D051841:Transcription Factor RelB; D000097782:NF-kappaB-Inducing Kinase