Characterization of the bovine IκBkinases(IKK)α and IKKβ, the regulatory subunit NEMO and their substrate IκBα

Abstract

The Nuclear factor (NF)-κB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-κB kinase) complex is a crucial step and a point of convergence of all known NF-κB signalling pathways. To analyse bovine IKKα (IKK1), IKKβ (IKK2) and IKKγ (or NF-κB Essential MOdulator, NEMO) and their substrate IκBα (InhibitorofNF-κB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IκBα are evolutionarily highly conserved components of the NF-κB pathway. Bovine IKKα and IKKβ are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKα, IKKβ, NEMO and the interaction of IKK with its substrate IκBα could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein–protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKα and IKKβ, also strongly interact with each other.