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Description
First described in B-lymphocytes, the canonical NF-κB signaling pathway is a ubiquitous pro-inflammatory, cell survival program that can be activated by a diverse array of cytokines, growth factors, bacterial, viral, and environmental stimuli. In resting cells the transcription factor, NF-κB, is sequestered in the cytosol by its association with inhibitor of κB (IκB) proteins. Upon induction by extracellular stimuli, IκB is phosphorylated at two N-terminal serines that target it for ubiquitylation and subsequent proteolytic degradation. NF-κB is thus freed to translocate to the nucleus where it enhances the transcription of a suite of target genes promoting cell growth and survival. The critical IκB phosphorylation event triggering NF-κB activation is performed by the inhibitor of κB kinase (IKK) complex. The IKK complex is composed of two highly conserved catalytic subunits, IKKα or IKK1 and IKKβ or IKK2, and a third essential, non-catalytic scaffolding subunit called IKKγ or NEMO. Activation of the catalytic subunits involves phosphorylation of two activation loop serines (176 and 180 for IKK1 and 177 and 181 for IKK2). This requires NEMO in vivo to articulate upstream factors to IKK activation. In vitro, however, overexpression and purification of IKK2 alone can produce an active kinase capable of trans auto-phosphorylation. As such it is not clear whether an upstream IKK kinase such as TAK1 is always required for IKK activation and/or if the inherent trans auto-phosphorylation activity of IKK plays a role. In this study, we have crystallized and solved a low resolution, 4 Å, X-ray structure of human IKK2 in an active conformation. Analysis of the asymmetric unit reveals arrays of "open" IKK2 dimers associating through two novel protein-protein interfaces that appear to facilitate the positioning of kinase domains for efficient activation loop trans auto-phosphorylation. Mutations of residues designed to abrogate these novel protein-protein interfaces reduced both the amount of S177,181 phosphorylation and N-terminal IκB[alpha] phosphorylation in HEK293T transfected cells and in in vitro kinase assays. We propose a mechanism of IKK2 trans auto-phosphorylation by an oligomerization dependent pathway through novel, transient protein-protein interactions. This would explain both the rapid kinetics and signal amplification observed in cells.
