Ubiquitination and deubiquitination involved in cell cycle progression

Abstract

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes degradation of mitotic regulators. Activation of the APC/C requires either Cdc20 or Cdh1, together with phosphorylation. In chapter 2 of this thesis, we show that Polo-like kinase-1 (Plk1) is required for activation of Cdh1-dependent APC/C function during late mitosis. Plk1-depletion prevents dephoshorylation of Cdh1. We found that Plk1 mediates its effect on Cdh1 through phosphorylation of the phosphatase Cdc14A, which controls the phosphorylation state of Cdh1. We conclude that Plk1 controls APC/CCdh1 activity through hCdc14A, to ensure proper cell cycle progression. RASSF1C is a splice-variant of the Ras-association domain family gene. In chapter 3 we describe the identification of a consensus site for Plk1-dependent phosphorylation in RASSF1C. We show that Plk1 can phosphorylate RASSF1C in vitro and that the proteins can interact in vivo. Over-expression of an active form of Plk1 reduces RASSF1C protein levels, whereas depletion of Plk1 increases the protein level of RASSF1C. Importantly, active Plk1 interferes with DNA damage-induced JNK-activation. Our data suggest a novel role for Plk1 in negatively regulating RASSF1C to silence DNA damage responses. To examine possible roles for deubiquitinating enzymes (Dubs) in G2/M progression, we describe an RNAi-based screen in chapter 4. By analyzing cell cycle progression of transfected cells, we identified 6 Dubs that appear to have a function in G2/M. Dub3 and Usp35 seem to be required for mitotic entry. Usp25 and Usp30 appear to play a role in mitotic checkpoint-activity or APC/C-inhibition. Usp8 and Usp39 appear involved in the mitotic checkpoint and cytokinesis. We further focused on the phenotype induced by two independent shRNAs against Usp30. However, the observed mitotic phenotypes did not correlate with the relative reduction of Usp30 mRNA, indicating that off-target effects are involved in the Usp30-depletion phenotype. We confirmed other hits of the screen with independent RNA oligos. Thus, although we emphasize the risk of off-target effects, we confirmed the validity of our screen and identified Dubs with a role in G2/M. In chapter 5 we further investigate Dub3 and showthat Dub3-depletion results in a G2 arrest. This arrest can be overcome early in G2. However, checkpoint inhibition after a prolonged G2 arrest prevents mitotic entry in Dub3-depleted cells. We find that Plk1, a downstream target of the G2-checkpoint, can not be re-activated in Dub3-depleted cells after a prolonged, DNA damage-induced arrest. Altogether, our data indicate a function for Dub3 in mitotic entry and recovery after DNA damage, possibly through regulation of Plk1. Accurate chromosome segregation relies on the mitotic spindle checkpoint, which restricst activity of the APC/C until all chromosomes are bipolarly attached. In chapter 6 we describe Usp39 as a new factor required to maintain the spindle checkpoint and support cytokinesis. We show that Usp39 is deprived of Dub activity. However, consistent with a described role for Usp39 in mRNA processing, we observed reduction in Aurora B-mRNA levels after Usp39-depletion. Our observations suggest Usp39 to be involved in splicing of Aurora B and other mRNAs that are essential for spindle checkpoint function.