Binding to IkB prevents the NF-kB:IkB complex from translocating to the nucleus – effectively preventing transactivation of NF-kB target genes. In the classical pathway of NF-kB activation, e.g. upon stimulation TNF, signaling pathways lead to activation of the beta subunit of the IkB kinase (IKK) complex which then phosphorylates IkB proteins on two N-terminal serine residues. Phosphorylated IkBs are recognized by the ubiquitin ligase machinery, leading to their polyubiquitination and subsequent degradation, or processing in the case of p100, by the proteasome.  Free NF-kB dimers translocate to the nucleus where they bind to specific sequences in the promoter or enhancer regions of target genes. Activated NF-kB can then be down-regulated through multiple mechanisms including the well-characterized feedback pathway whereby newly synthesized IkBa protein binds to nuclear NF-kB and exports it out to the cytosol.

 

There are seven IkB family members - IkBa, IkBb, BCL-3, IkBe, IkBg and the precursor proteins p100 and p105 – that are characterized by the presence of five to seven ankyrin repeats that assemble into elongated cylinders that bind the dimerization domain of NF-kB dimers. Structures reveal that the IkB proteins mask the nuclear localization sequence (NLS) of p65, leading to steady-state cytosolic localization. The balance between cytosolic and nuclear localization is altered upon IkBa degradation since it removes the contribution of the IkB nuclear export sequence (NES) and exposes the masked NLS of p65, resulting in predominantly nuclear localization of NF-kB.

Degradation of IkB is a tightly regulated event that is initiated upon specific phosphorylation by activated IKK. The IKK activity in cells can be purified as a 700 – 900 kDa complex, and has been shown to contain two kinase subunits, IKKa (IKK1) and IKKb (IKK2), and a regulatory subunit NEMO (NF-kB essential modifier) or IKKg.  In the classical NF-kB signaling pathway, IKKb is both necessary and sufficient for phosphorylation of IkBa on Ser32 and Ser36, and IkBb on Ser19 and Ser23.

Upon phosphorylation by IKKs, IkB proteins are recognized and ubiquitinated by members of the Skp1-Culin-Roc1/Rbx1/Hrt-1-F-box (SCF or SCRF) family of ubiquitin ligases. bTrCP (E3RS or Fbw1a), the receptor subunit of the SCF family ubiquitin ligase machinery, binds directly to the phosphorylated E3 recognition sequence (DS*GXXS*) on IkB. Recognition of IkB leads to polyubiquitination at the conserved residues, Lys21 and Lys22 on IkBa, by the E3 SCF-bTrCP and the E2 UbcH5.