The K48-K63 Branched Ubiquitin Chain Regulates NF-κB Signaling

Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1β, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.