The covalent attachment of ubiquitin to a protein controls a range of cellular processes in eukaryotes by altering the protein's stability, localization, or activity. Ubiquitylation requires the activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). This enzymatic cascade modifies substrates with either a single ubiquitin or polyubiquitin chains linked through lysine residues of ubiquitin. Because ubiquitin contains seven lysines, different chain types are possible, and chain topology determines the fate of the modified protein. For example, Lys48-linked chains target proteins to the proteasome, while non-proteolytic Lys63 chains are involved in complex reorganization and signaling pathways. Although the physiological roles and formation of these chain types has been extensively studied, the characterization of other chain types is lacking. In this dissertation, I present work describing the biological function and assembly of Lys11-linked ubiquitin chains.
Lys11-linked chains are formed on substrates of the human anaphase-promoting complex (APC/C), an E3 ligase essential for proliferation, promoting their degradation by the proteasome. Because this chain type was proposed to be essential for regulating mitosis, we undertook an extensive investigation into how Lys11 chains are build by the APC/C, with a focus on the role of E2 enzymes. In the Chapters below, we report that APC/C-dependent formation of Lys11- linked chains requires the activity of two E2 enzymes: a chain-initiating E2 Ube2C and a Lys11- specific, chain-elongating E2 Ube2S. These E2s likely bind the APC/C at the same time, representing a unique way for the APC/C to achieve processivity.
In most instances, linkage-specific ubiquitin chain assembly is promoted by E2s, yet this process has only been well characterized for Lys63-linked chains. Therefore, we next investigated the molecular mechanism underlying the Lys11-specificity of Ube2S. We report that Ube2S engages in essential non-covalent interactions with ubiquitin that are required for processive and specific ubiquitylation. Lys11-specificity is generated by substrate-assisted catalysis, in which an acidic residue of the substrate ubiquitin is essential for generating a catalytically competent active site.
To solidify the role of Lys11-linked chains and the APC/C in mitotic control, we developed a Lys11-specific antibody. Using this antibody, we show that the level of Lys11 chains in HeLa cells increases dramatically as cells pass through mitosis, while the level of Lys48 chains remains constant. Importantly, knockdown of the Ube2C/Ube2S E2 module inhibits formation of Lys11 chains, suggesting that the APC/C and its E2s are the major enzymatic machinery responsible for forming K11 chains in mitosis.