| The transcription factor NF-κB plays a key role in mediating immune and inflammatory responses in higher organisms. In unstimulated cells, NF-κB is sequestered in an inactive complex by one of the inhibitory molecules of the IκB family of proteins, such as IκBα. The activation of NF-κB involves the controlled phosphorylation, ubiquitination, and subsequent degradation of these inhibitory proteins. The key regulatory step in this process is the phosphorylation of IκBα on two specific serines in the amino terminus, S32 and S36, by a 700 kDa IκB kinase complex, containing two catalytic components, IKKα and IKKβ, as well as a third, structural component, IKKγ.;I demonstrate that IKKα/β can directly phosphorylate the critical serines in two IκB family members, IκBα and IκBβ. I also show that the 700 kDa MEKK1-inducible ubiquitin-inducible IκB kinase complex identified by our lab contains both IKKα and IKKβ, thus linking the work performed on this complex to these direct IκB kinases. My work further supports a role for a putative upstream kinase of this complex, MEKK1.;I also report the identification and characterization of a novel PMA-inducible IκB kinase complex, distinct from the IKKα/β/γ complex. I have characterized one kinase from this complex, which I designate IKKϵ, that is homologous to IKKα/β. Though recombinant IKKϵ directly phosphorylates only serine 36 of IκBα, the PMA-activated endogenous IKKϵ complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant negative mutant of IKKϵ blocks induction of NF-κB by both PMA and activation of the T cell receptor, but has no effect on the activation by TNFα or IL-1. These observations indicate that the activation of NF-κB requires multiple distinct IκB kinases, which respond to both overlapping and discrete signaling pathways.;Finally, I report the partial purification and characterization of a newly identified kinase closely related to IKKϵ, designated TBK1. I demonstrate that this kinase also specifically phosphorylates S36 of IκBα, and similar to IKKϵ, is not responsive to certain inducers of the IKKα/β/γ complex. |
