NFkB Activating Kinase, active, Recombinant, Human (Nuclear Factor kappa B, TANK-Binding Kinase 1, NAK, TBK1)

N-terminal His6-tagged, recombinant, full length, human TBK1, expressed by baculovirus in Sf21 cells.\n\nThe activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB.\n\nSpecific Activity: \n~300U/mg, where one unit of TBK1 activity is defined as 1nmol phosphate incorporated into 200uM CK1tide (KRRRAL[pS]VASLPGL) per minute at 30 degrees C with a final ATP concentration of 100uM.\n\nApplications:\nMS Tryptic Fingerprint: Confirmed identity as TBK1 with 50% amino acid coverage of the translated native sequence.\nTBK1 Kinase Assay: 3.97-50ng of this lot of enzyme phosphorylated 200uM CK1tide in the assay. Assay background was subtracted from the actual counts to yield the results.\n\nStorage and Stability:\nFor long-term storage, store at -70 degrees C. Aliquots are stable for at least 6 months at -70 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer

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SPECIFICATIONS

Catalog Number

N2301-40D

Size

10ug

Form

Supplied as a liquid in 50mM Tris-HCl, pH 7.5, 300mM sodium chloride, 0.1mM EGTA, 0.03% Brij-35, 270mM sucrose, 1mM benzamidine, 0.2mM PMSF and 0.1% 2-mercaptoethanol.

Purity

Purified using Ni2+/NTA agarose. ~42% by SDS PAGE and Coomassie blue staining.

References

1. Pomerantz, J.L. & Baltimore, D., EMBO J. 18(23): 6694-6704 (1999).