Biochemical Mechanism of Protein Kinase Activation by the Ubiquitination System

The NF-κB signaling pathway is important for immune, inflammatory and stress responses of cells and can be activated by a variety of extracellular stimuli. In the IL1R/TLR signaling pathway, NF-κB is activated through activation of TAK1-IKK cascade by TRAF6 and Ubc13/Uev1A in a polyubiquitinationdependent manner. Mechanistically how TAK1 is activated by the TRAF6- Ubc13/Uev1A dependent polyubiquitination system is unknown. Whether TAK1 and IKK kinases can be activated by other than TRAF6-Ubc13/Uev1A is an open question. By inactivating ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2 using NEM to further dissect the system, studies on how TAK1 is activated by the TRAF6-Ubc13/Uev1A system have revealed that 1) polyubiquitination step and kinase activation step can be un-coupled; 2) Polyubiquitination step generates unanchored K63-linked polyubiquitin chains as the kinase activators; 3) Coiled-coil domain of TRAF6 is required for synthesis of active polyubiquitin chains. Through biochemical fractionation, I purified UbcH5 as another E2 that works with TRAF6 to directly activate IKK. Mechanistic analysis on how TRAF6/UbcH5 activates IKK reveals that TRAF6-UbcH5 synthesize mixedlinkage- linked unanchored polyubiquitin chains and this polyubiquitin chains function as direct activators for IKK activation.