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Description
NF-_B is a master transcription factor that elevates the expression of genes involved in arthritis, asthma, autoimmune disease, and cancer. NF-_B exists as part of an inactive complex in virtually all mammalian cells types. Induction of NF-_B activity results from any of a vast number of stress signals. The central integrator of all these diverse signaling pathways is the Inhibitor of _B Kinase (IKK), a catalytically active kinase complex that resides directly upstream of NF-_B and is responsible for directly responsible for its activation. IKK is composed of three polypeptide subunits. Two of these, IKK_ and IKK_, contain functioning kinase catalytic domains while the third, NEMO, functions as a necessary regulatory subunit. The IKK_ subunit is chiefly responsible for NF-_B activation in response to pro-inflammatory stimuli. However, IKK_ also phosphorylates other substrates unrelated to the NF-_B pathway. Therefore, drugs designed to target the kinase activity of IKK_ would likely affect other signaling pathways. Recent studies have revealed that NF-_B activation by IKK first requires autophosphorylation by IKK_ on its tyrosine residues 188 and 199. In order to address this as well as fundamental biophysical questions regarding the structure of functioning IKK complexes, we have endeavored to develop protocols for milligram-scale expression and purification of a human multi-subunit IKK complexes and an assay that can distinguish between the tyrosine kinase auto-catalytic activity of IKK_ and its serine-specific NF-_B inducing state. To these ends we designed and tested several systems involving recombinant baculoviruses that encode for IKK subunits individually or in tandem. We find that, unlike in the case of the homologous IKK complex from Drosophila melanogaster, the human IKK complex fails to spontaneously assemble either when co-expressed simultaneously in the same insect cell suspension or when introduced separately after expression in either insect cells or E. coli bacteria. This finding seriously limits our ability to carry out structural studies with mammalian IKK complexes. A modified approach, using the Multi-Bac system, is proposed for future efforts at producing this material. In a separate pilot study, anti-phospho-Tyrosine and -Serine antibodies were employed to simultaneously detect the two IKK_ catalytic specificities required for NF-_B activation. The addition of the general kinase inhibitor staurosporine and simultaneous fluorescence and immunodetection confirm that these two catalytic specificities can be monitored independently and suggest that an assay based on this principle might serve to identify small molecule inhibitors that are uniquely specific for the NF-_B-activating conformation of IKK_..
