Phosphoproteome dynamics during mitotic exit in budding yeast

Abstract

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.

Mitotic exit is controlled during anaphase by an Aurora B-Cyclin B1/Cdk1 crosstalk

“…Most importantly, we show that Cyclin B1 homeostasis during anaphase relies on both Aurora B-mediated phosphorylation and localization at the spindle midzone. In concert with previous work , our findings unveil an unexpected crosstalk between molecular "rulers" inactivation promotes the recruitment of PP1 phosphatase to chromosomes to locally oppose Aurora B phosphorylation (Qian et al, 2015) and recent findings in budding yeast demonstrating equivalent phosphorylation and dephosphorylation events during mitotic exit (Touati et al, 2018). Moreover, it provides an explanation for the coordinated action of two unrelated protein kinases that likely regulate multiple substrates required for mitotic exit (Afonso et al, 2017;Kettenbach et al, 2011;Petrone et al, 2016).…”

In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

“…3c). Despite having less temporal resolution than more focused investigations of mitotic-specific phosphorylation dynamics 25 , to our knowledge this analysis currently represents the most extensive catalog of cell cycle-dependent phosphorylation events for this organism at various cell cycle stages. Figure 2 was used for this analysis.…”

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

“…Phactr1 expression was induced by doxycycline addition to 1 μg/ml for 5 hr. Cells were harvested processed for mass spectrometry essentially as described ( Pattison et al, 2016 ; Touati et al, 2018 ) with minor modifications. 1 mg each of ‘light’ and ‘heavy’ labelled lysates were mixed and dried.…”