Abstract
Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.
Highlights
Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate
Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain
Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways
Summary
One requirement for understanding the biochemical activities associated with an E3 is to determine the identity of the relevant E2 or a subset of E2s that is capable of cooperating with the E3 in question. We could resolve up to six distinct ubiquitinated intermediates in the presence of the Lys63-only Ub mutant, and the accumulation of high molecular weight reaction products Ͼ75 kDa (corresponding to Ub5-n in Fig. 4C) was effectively abolished These observations suggest that specificity for Lys-63-linked Ub chain synthesis on WBP2CTK222 is a function of chain length and that chains are elongated by WWP1 in a manner that consists of mixed linkages once the chain reaches four Ub subunits in length. Reaction products were quenched in 8 M urea at the indicated times, WBP2CTK222 species were purified under denaturing conditions, and chain linkages were analyzed by LC-MS/MS The results of this analysis clearly demonstrated that specificity for Lys-63-linked Ub chain synthesis by WWP1 was reduced as chain length increased (Fig. 7, A and B). These results provide direct evidence for a branching activity by WWP1 and demonstrate that WWP1 is capable of extending Ub chains of complex topology and multiple linkages off of a homogeneous Lys-63-linked tetraubiquitin chain
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