Abstract
Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.
Highlights
IB kinase  is a key regulator in the NB signaling pathway
Protein Expression and Purification—A construct encoding a hexahistidine tag followed by residues 1– 664 of hIKK was cloned into a pBacPAK vector (Clontech) to enable its expression in baculovirus-infected insect cells
Full-length hIKK was heterogeneously phosphorylated at 6 –19 sites owing to inclusion of the C-terminal NEMO binding domain region, which is prone to heavy phosphorylation
Summary
IB kinase  is a key regulator in the NB signaling pathway. Results: Crystal structure of a human IKK asymmetric dimer shows one kinase active site phosphorylated and in the active conformation and the other unphosphorylated and inactive. Sis, asthma, arthritis, cancer, cachexia, diabetes, euthyroid sick syndrome, acquired immune deficiency syndrome, inflammatory bowel disease, and stroke [3, 4] In their resting or inactive state, NF-B factors exist as cytoplasmic complexes with members of the NF-B inhibitor protein family, IB␣, IB, and IB⑀. Phosphorylation permits IB proteins to be polyubiquitinated and catabolized by the proteasome Liberation from their inhibitors leaves NF-B factors free to enter the nucleus and activate transcription of genes encoding proteins that participate in the immune and inflammatory response, cell adhesion, growth control, and protection against apoptosis. NEMO is essential for the activation and substrate specificity of IKK [9, 10] Both IKK␣ and IKK contain an N-terminal kinase domain (KD, residues 15–308 in hIKK), a leucine zipper region (residues 458 – 479), and a helix-loop-helix region (residues 603– 642) [11]. We complement our data with biochemical analysis of the dimer formation and mass spectrometric analysis of the phosphorylation state
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