Abstract
Canonical activation of NF-kappa B is mediated via phosphorylation of the inhibitory I kappa B proteins by the I kappa B kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKK alpha and IKK beta subunits and a presumed regulatory protein termed NEMO (NF-kappa B essential modulator) or IKK gamma. NEMO/IKK gamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappa B activator) as a NEMO/IKK gamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappa B activation in a complex with TANK-binding kinase 1 (TBK1) or IKK epsilon, two kinases distantly related to IKK alpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKK epsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKK gamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKK epsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappa B activation.
Highlights
Canonical activation of NF-B is mediated via phosphorylation of the inhibitory IB proteins by the IB kinase complex (IKK)
These results indicate that TANK can synergize with IKK⑀ or TANK-binding kinase 1 (TBK1) to link them to IKK complexes, where the two kinases may modulate aspects of NF-B activation
TBK1 and IKK⑀ were PCR-amplified from a liver cDNA library. cDNAs encoding TANK and IKK⑀ were cloned into pcDNA3.1 FLAG (Invitrogen) for expression in mammalian cells, and TBK1 and IKK⑀ were cloned into pcDNA3.1 Myc
Summary
Vol 277, No 40, Issue of October 4, pp. 37029 –37036, 2002 Printed in U.S.A. Association of the Adaptor TANK with the IB Kinase (IKK) Regulator NEMO Connects IKK Complexes with IKK⑀ and TBK1 Kinases*. It has been demonstrated that the IKK kinases target the so-called small IB inhibitors, of which the IB␣ is the prototype, but that they phosphorylate and regulate the p105/NF-B1 and p100/NF-B2 precursors, leading either to their proteolytic degradation or to their processing to p50 and p52, respectively [15,16,17,18] In addition to these functions, IKK kinase activity may modulate the transactivation potential of the NF-B proteins liberated by the degradation of the inhibitors; activated IKK kinases have been shown to phosphorylate a transactivation domain of RelA, thereby promoting its ability to transcriptionally transactivate genes [19]. We provide evidence that the TANK-binding domain of NEMO may be important in transmitting signals
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