Direct activation of protein kinases by unanchored polyubiquitin chains

Abstract

The nuclear factor κ enhancer binding protein (NF-κB) signalling pathway is important for a range of cellular processes including immune function. Here Xia et al. show that free Lys63 polyubiquitin chains generated, which are not linked to any protein substrates, can directly activate kinases in the NK- κB signalling pathway. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented kinase activation. These results suggest that unanchored polyubiquitin chains much like second messengers can directly activate kinases in immune and inflammatory pathways. The ubiquitin ligase TRAF6 is essential for the activation of NF-κB and MAP kinases in several signalling pathways important for a range of cellular processes including immune function. TRAF6 functions together with a ubiquitin-conjugating enzyme complex to activate the TAK1 kinase complex downstream, which in turn leads to the activation of NF-κB. Here, by reconstituting TAK1 activation in vitro, free Lys 63 polyubiquitin chains are shown to activate TAK1 directly. TRAF6 is a ubiquitin ligase that is essential for the activation of NF-κB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors1,2,3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of UBC13 (also known as UBE2N) and UEV1A (UBE2V1) to catalyse Lys 63-linked polyubiquitination, which activates the TAK1 (also known as MAP3K7) kinase complex4,5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to the activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL1R and Toll-like receptor pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. By reconstituting TAK1 activation in vitro using purified proteins, here we show that free Lys 63 polyubiquitin chains, which are not conjugated to any target protein, directly activate TAK1 by binding to the ubiquitin receptor TAB2 (also known as MAP3K7IP2). This binding leads to autophosphorylation and activation of TAK1. Furthermore, we found that unanchored polyubiquitin chains synthesized by TRAF6 and UBCH5C (also known as UBE2D3) activate 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 new mechanism of protein kinase regulation.