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Dual mechanism for APC/C activation by integrated ubiquitylation and dephosphorylation

  • Author(s): Craney, Allison Christine
  • Advisor(s): Rape, Michael
  • et al.
Abstract

The Anaphase Promoting Complex (APC/C) is a multi-subunit ubiquitin ligase that is essential for cell cycle progression. Ube2S and Ube2C are dedicated APC/C E2-conjuating enzymes that build ubiquitin chains upon mitotic substrates. Two critical outcomes exist for this activity. First, proteins that have fulfilled their mitotic roles are removed by proteasomal degradation. Second, ubiquitylation is required to disrupt inhibitory binding of the mitotic checkpoint complex (MCC) to APC/C through the promotion of CDC20 degradation, thereby silencing the spindle assembly checkpoint (SAC). Surprisingly, however, depletion of Ube2S and Ube2C does not prevent mitotic exit. We decided to perform a Ube2S genetic interaction screen in order to better understand the functional importance of the E2 enzymes in mitosis. We discovered that Ube2S activity is essential for cell survival when one of several regulators of kinetochore-microtubule dynamics is depleted, such as the mitotic kinesin Kif18A. Co-depletion of Ube2S with one or more of these regulators results in a prolonged or permanent prometaphase arrest dependent upon the SAC. These data suggest a role for Ube2S and the APC/C in prometaphase, a time in which APC/C activity has been thought to be low. Further analysis of Ube2S in prometaphase has revealed that Ube2S contributes heavily to proper oscillatory movement of kinetochores. We also examined changes in APC/C composition when its ubiquitylation activity had been compromised, to mimic Ube2S depletion. We found that when APC/C activity is limited in prometaphase, phosphatases interact with APC/C in a Cdc20-dependent manner. Furthermore, removal or depletion of phosphatase activity results in decreased ubiquitylation of APC/C substrates and subsequent stabilization of proteins such as geminin. Reduced APC/C activity when dephosphorylation is blocked is likely due to less activity of Ube2S and to loss of co-activator function by CDC20. Taken together, these data have identified a necessary, integrated function for ubiquitylation and phosphatase activity in the regulation of cell division.

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