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Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin–CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin–CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4–6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

Multiple protein kinases regulate cell-cycle progression, of which the cyclin-dependent kinases (CDKs) are thought to act as upstream master regulators. We have used quantitative phosphoproteomics to analyze the fission yeast cell cycle at sufficiently high temporal resolution to distinguish fine-grain differences in substrate phosphorylation dynamics on a proteome-wide scale. This dataset provides a useful resource for investigating the regulatory dynamics of cell-cycle kinases and their substrates. For example, our analysis indicates that the substrates of different mitotic kinases (CDK, NIMA-related, Polo-like, and Aurora) are phosphorylated in sequential, kinase-specific waves during mitosis. Phosphoproteomics analysis after chemical-genetic manipulation of CDK activity suggests that the timing of these waves is established by the differential dependency of the downstream kinases on upstream CDK. We have also examined the temporal organization of phosphorylation during G1/S, as well as the coordination between the NDR-related kinase Orb6, which controls polarized growth, and other cell-cycle kinases.

Avian predation on juvenile salmonids is an important source of mortality in freshwater and estuarine habitats when birds and salmonids overlap spatially and temporally. We assessed the potential impact of avian predation upon juvenile steelhead Oncorhynchus mykissin a coastal watershed in central California. We conducted stream surveys between 2008 and 2010 to determine the composition, distribution, and density of piscivorous birds in areas that provide rearing habitat for juvenile steelhead. The most commonly sighted bird species were common mergansers Mergus merganserand belted kingfishers Megacyrle alcyon. The density of avian predators varied spatially and temporally but was greatest in the estuary regardless of season and decreased with increasing distance from the estuary. In the absence of local predator diet data, we applied a bioenergetics model to estimate the potential predation on juvenile steelhead by mergansers and kingfishers in the Scott Creek estuary. Model parameters included (1) published values of bird energetic requirements and steelhead energy density, (2) the number of birds present in the estuary during the closure period (from stream surveys), and (3) the size frequency and abundance of steelhead present in the estuary during closure. We predicted the extent of predation for different values of steelhead in bird diets, accounting for uncertainty in the estimates using a Monte Carlo simulation approach. With the assumed contribution of steelhead to the diet ranging from 20% to 100%, the population of kingfishers foraging in the Scott Creek estuary had the potential to remove 3–17% of annual production, whereas mergansers had the potential to remove 5–54% of annual steelhead production. Our results suggest that predation by avian species, particularly mergansers, is an important source of mortality for threatened steelhead populations in central California and should be addressed in future salmonid research and recovery planning. Received February 13, 2013; accepted June 4, 2013

Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS). We have used this technology to label 20 Saccharomyces cerevisiaesamples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological and mass spectrometry sample preparation and analysis. For complete details on the use and execution of this protocol, please refer to Touati et al. (2019).

Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during exit from mitosis. In budding yeast, the Cdc14 phosphatase is thought to be a major regulator at this time, while in higher eukaryotes PP2A phosphatases take a dominant role. Here, we use time-resolved phosphoproteome analysis in budding yeast to evaluate the respective contributions of Cdc14, PP2ACdc55, and PP2ARts1. This reveals an overlapping requirement for all three phosphatases during mitotic progression. Our time-resolved phosphoproteome resource reveals how Cdc14 instructs the sequential pattern of phosphorylation changes, in part through preferential recognition of serine-based cyclin-dependent kinase (Cdk) substrates. PP2ACdc55and PP2ARts1in turn exhibit a broad substrate spectrum with some selectivity for phosphothreonines and a role for PP2ARts1in sustaining Aurora kinase activity. These results illustrate synergy and coordination between phosphatases as they orchestrate phosphoproteome dynamics during mitotic progression.