Methods to assess the activation of the alternative (noncanonical) NF-κB pathway by non-death TNF receptors

Methods Mol Biol. 2015:1280:103-19. doi: 10.1007/978-1-4939-2422-6_7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / pharmacology
  • Electrophoretic Mobility Shift Assay
  • Enzyme Activation
  • Enzyme Stability
  • Humans
  • I-kappa B Kinase / metabolism
  • Inhibitor of Apoptosis Proteins / metabolism
  • Ligands
  • NF-kappa B / metabolism*
  • NF-kappa B p52 Subunit / metabolism
  • NF-kappaB-Inducing Kinase
  • Phosphorylation
  • Protein Binding
  • Protein Serine-Threonine Kinases / metabolism
  • Protein Transport
  • Proteolysis
  • Receptors, Tumor Necrosis Factor / agonists
  • Receptors, Tumor Necrosis Factor / metabolism*
  • Signal Transduction* / drug effects
  • TNF Receptor-Associated Factor 2 / metabolism
  • TNF Receptor-Associated Factor 3 / metabolism
  • Transcription Factor RelB / metabolism

Substances

  • Antibodies, Monoclonal
  • Inhibitor of Apoptosis Proteins
  • Ligands
  • NF-kappa B
  • NF-kappa B p52 Subunit
  • Receptors, Tumor Necrosis Factor
  • TNF Receptor-Associated Factor 2
  • TNF Receptor-Associated Factor 3
  • Transcription Factor RelB
  • Protein Serine-Threonine Kinases
  • I-kappa B Kinase