Methods to Assess the Activation of the Alternative (Noncanonical) NF-kappaB Pathway by Non-death TNF Receptors.

The alternative or noncanonical NF-kappaB pathway regulates the generation of p52-containing NF-kappaB 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-kappaB-Inducing Kinase) and IKKalpha (Inhibitor of kappaB Kinase alpha). These kinases create a phosphodegron for the E3 ligase SCF-beta-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-kappaB pathway. These methods uncover cytosolic and nuclear biochemical modifications of key proteins of the alternative NF-kappaB pathway required prior to the transcription of NF-kappaB target genes.