Direct Activation of Protein Kinases by Unanchored Polyubiquitin Chains

Abstract

TRAF6 is a ubiquitin ligase essential for the activation of NF-κB and MAP kinases in multiple signaling pathways including those emanating from the interleukin-1 and Toll-like receptors (IL-1R/TLR) 1- 3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A to catalyze Lys-63 (K63)-linked polyubiquitination, which activates the TAK1 kinase complex 4, 5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL-1R/TLR pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. Herein, we reconstituted TAK1 activation in vitro using purified proteins and found that free K63 polyubiquitin chains, which are not conjugated to any target protein, directly activated TAK1 through binding to the ubiquitin receptor TAB2. This binding leads to autophosphorylation and activation of TAK1. We also found that unanchored polyubiquitin chains synthesized by TRAF6 and Ubc5 activated the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a novel mechanism of protein kinase regulation.