- Paper Report
- Open Access
TANK-binding kinase 1 activates NF-?B
- Andy Finch1
© Current Science Ltd 2000
- Published: 13 January 2000
The involvement of the tumour necrosis factor receptor (TNFR)-associated factor (TRAF) family of proteins in NF-?B and c-Jun amino-terminal kinase (JNK) activation by pro-inflammatory cytokines has been well documented. NF-?B activation by TRAFs occurs through NF-?B-inducing kinase (NIK), and the I?B kinase (IKK), leading to I?B phosphorylation and degradation. TRAF associated NF-?B activator (TANK) is a TRAF-binding protein which can activate NF-?B in a TRAF-dependent fashion yet inhibits NF-?B activation by pro-inflammatory cytokines. This article describes the identification of a TANK-binding kinase, TBK1, which mediates the TANK/TRAF-dependent activation of NF-?B and proposes that TANK and TBK1 can compete with pro-inflammatory receptor complexes in the activation of NF-?B. To clarify the seemingly contradictory role of TANK in the NF-?B pathway by identifying and characterising other molecules which interact with TANK.
Transfection of dominant-negative components of the TRAF/NF-?B pathway inhibited NF-?B activation by a constitutively active N-terminal portion of TANK (TANK 1-190), suggesting that the activation of NF-?B by TANK proceeds via TRAF2, NIK and the IKKs. A yeast two-hybrid screen, using TANK 1-190 as bait yielded TBK1, which could activate NF-?B when overexpressed: the dominant-negative form of TBK1 could block NF-?B activation by TANK, and NF-?B activation by TBK1 was also dependent on NIK and the IKKs as well as on TRAF2.
Co-precipitation of TRAF2, TBK1 and TANK (full length and 1-190) was demonstrated, indicating that these proteins can form a ternary complex. Cotransfection of an N-terminal mutant of TANK (1-168, which cannot bind TRAFs) prevented TBK1-induced NF-?B activity. Thus the ternary complex formed between these proteins seems to be necessary for TBK1-mediated activation of NF-?B. TBK1 transfection caused phosphorylation of TANK and TRAF2, although this was not necessary for complex formation.
Importantly, TBK1 did not inhibit TNF/IL-1/CD40L-induced activation of NF-?B (indicating that it lies on a distinct pathway) and it was able to overcome the ability of full length TANK to inhibit CD40L-induced NF-?B activity (indicating that TANK interacts with both pathways). Finally, a dominant-negative TBK1 had no effect on the TANK/TRAF2-induced activation of JNK.
Methods used included identification of TBK1 by yeast two-hybrid screening, co-transfection of wild-type and mutant forms of NF-?B pathway members with NF-?B reporters and co-immunoprecipitation of TRAF2/TANK/TBK1. Also, in vitro kinase assays for JNK activity and electrophoretic gel mobility shift assays (EMSA) for NF-?B activity were performed. The signalling studies were performed in 293 cells.