K63 ubiquitylation triggers proteasomal degradation by seeding branched ubiquitin chains

Abstract

Different polyubiquitin chain linkages direct substrates toward distinct cellular pathways. K63-linked ubiquitylation is known to regulate proteasome-independent events such as signal transduction, but its function in the context of heterogeneous ubiquitin chains remains unclear. Here, we report that K63 ubiquitylation plays a critical role in proteasome-mediated substrate degradation by serving as a “seed” for K48/K63 branched ubiquitin chains. Quantitative analysis revealed that K48/K63 branched linkages preferentially associate with proteasomes in cells. We found that ITCH-dependent K63 ubiquitylation of the proapoptotic regulator TXNIP triggered subsequent assembly of K48/K63 branched chains by recruiting ubiquitin-interacting ligases such as UBR5, leading to TXNIP degradation. These results reveal a role for K63 chains as a substrate-specific mark for proteasomal degradation involved in regulating cell fate. Our findings provide insight into how cellular interpretation of the ubiquitin code is altered by combinations of ubiquitin linkages. Significance Posttranslational modification of proteins by ubiquitylation regulates numerous biological processes. Polyubiquitin chain linkages are critical determinants that direct substrates to distinct pathways, a concept referred to as the ubiquitin code. Lysine-63 (K63)-linked polyubiquitin chains are the second most abundant linkage type in cells, and are known to regulate proteasome-independent pathways such as signal transduction and endocytosis. We report that K63-linked polyubiquitylation of the proapoptotic regulator TXNIP by the ubiquitin ligase ITCH triggers proteasome-mediated protein degradation. K63 ubiquitin chains facilitate subsequent assembly of K48/K63 branched ubiquitin chains through recruitment of ubiquitin ligases assembling K48 chains, thereby directing substrates to the proteasome. These results reveal an unappreciated ubiquitin-dependent pathway leading to the proteasome.