Mechanisms Regulating Ubiquitin Chain Formation during Mitosis

The small protein ubiquitin is a post-translational modification that is essential for regulating many cellular processes in eukaryotes. In a series of enzymatic steps, ubiquitin is activated by the ubiquitin-activating enzyme E1 and then transferred to the active site of an E2 ubiquitin-conjugating enzyme. An E3 ubiquitin ligase interacts with the ubiquitin-charged E2 and the substrate protein to facilitate transfer of ubiquitin to a substrate residue, typically lysine. Ubiquitin modifications can affect substrate protein interactions, localization, or stability. With such versatile outcomes, ubiquitylation regulates countless cellular processes, including immune signaling, transcriptional regulation, DNA damage response, and the cell cycle. The Anaphase-Promoting Complex (APC/C) is a RING domain-containing E3 ligase that regulates mitotic progression by building ubiquitin chains on its cell cycle regulator substrates, targeting them for proteasomal degradation. APC/C works with two E2 enzymes to first initiate substrate ubiquitylation using Ube2C and then to build ubiquitin chains with Ube2S. How APC/C coordinates the activity of these E2 enzymes to processively build chains on substrates is not well understood. Additionally, it not known how these E2s contribute to mitotic progression: while APC/C is essential for mitotic exit, depletion of its cognate E2 enzymes does not have dramatic effects. In this dissertation, I present work determining how APC/C works with its E2s to build ubiquitin chains and how this is important for regulating cell cycle progression./nAPC/C ubiquitylates numerous cell cycle regulators at particular times in mitosis to control spindle assembly, inactivation of the Spindle Assembly Checkpoint, and mitotic exit. While APC/C interacts with both substrates and with E2 enzymes at defined interfaces, it is unclear how chain formation occurs. In order to add a new ubiquitin molecule to the growing chain, ubiquitin-charged Ube2S must be in close proximity to the distal end of the chain. In Chapter 2, we report that APC/C stimulates chain formation by increasing the affinity between Ube2S and an acceptor ubiquitin at the end of the substrate-conjugated chain. Our finding support a model in which APC/C tethers the end of the growing chain close to the E2 active site to promote efficient chain formation without adjusting interactions with substrate or E2./nTo further understand how APC/C-specific E2 enzymes regulate mitosis, we investigated the role of Ube2S in governing APC/C activity at the significant transition from early mitosis with active Spindle Assembly Checkpoint signaling to late mitosis and mitotic exit. In Chapter 3, we found that Ube2S affects dynamics of the spindle checkpoint and is found in mitotic checkpoint complexes with the phosphatase PP2AB56, which has a well-described role in stabilizing kinetochore-microtubule attachments and turning off the checkpoint. We report that PP2AB56 promotes ubiquitin chain formation by APC/C, suggesting that there is collaboration between phosphatase and ubiquitylation activities to couple spindle checkpoint inaction with APC/C activation.

Advisor: Rape, Michael