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Summary
First detailed phosphoproteomic analysis of cyclin-dependent kinase (CDK) substrates
A single fused cyclin-CDK can drive the cell cycle and direct temporal CDK substrate phosphorylation
Regulation is quantitative, not qualitative: phosphorylation timing and hence the order of cell cycle events are mostly set by rising levels of CDK activity
A CDK-dependent kinase hierarchy leads to ordered kinase-specific phosphorylation waves during mitosis
CDK activity is partly regulated by compartmentalisation. The hydrophobic patch in cyclin B is required to localise CDK to the spindle pole body, where some of its most refractory mitotic substrates reside
In eukaryotic cells, progression from G1 into S phase and from G2 into mitosis (M) is regulated by a succession of cyclin-dependent kinase complexes, which are differentially expressed throughout the cell cycle. In contrast to higher eukaryotes, which have multiple cyclin-dependent kinases (CDKs), the fission yeast Schizosaccharomyces pombe contains only one, Cdc2, which can be activated by binding any of four mitotic cyclins or two further meiotic cyclins. This simpler system has been used for many years to address fundamental questions about basic principles of cell cycle regulation.
The textbook view of cell cycle control is that there are inherent differences in substrate specificity between different cyclin-CDKs, and it is the rise and fall of these cyclin-CDKs in waves of activity during the cell cycle that establishes and maintains temporal order: different subsets of critical substrates are phosphorylated as successive cyclin-CDK complexes form. However, this qualitative model has been overturned by recent work from Paul Nurse’s laboratory showing that in fission yeast, a quantitative mechanism, based on CDK activity thresholds, underpins CDK substrate phosphorylation and ordering of the cell cycle.
The qualitative view of cell cycle regulation was initially challenged by Coudreuse and Nurse (2010) when they demonstrated that in fission yeast, it was possible to delete Cdc2 and all the endogenous cyclins, and replace them with a single protein, Cdc13-L-Cdc2, in which Cdc2 was fused to the mitotic B-type cyclin Cdc13. Like Cdc13, the chimaeric protein (hereafter CDK) accumulated throughout the cell cycle and was degraded at the end of mitosis, and it could carry out all the events of both the meiotic and mitotic cell cycles (Gutiérrez-Escribano and Nurse, 2015).
Based on this genetic approach, Nurse and colleagues proposed a quantitative model for cell cycle control, in which a regular rise in kinase activity determines progression: low activity would bring about S phase, medium activity would block re-initiation of replication — important for maintaining ploidy — and the highest levels would orchestrate the onset of mitosis.
To provide biochemical evidence for this model, Nurse’s group turned to a systems analysis of CDK activity in fission yeast, collaborating with the Crick mass spectrometry facility to use phosphoproteomics to identify CDK substrates phosphorylated at different stages during the cell cycle (Swaffer et al, 2016). Using an ATP analogue-sensitive version of CDK, they synchronised cells in mitosis or S phase, inactivated CDK activity by adding the inhibitor 1-NmPP1, and found some 275 direct substrates by identifying immediate and continuous dephosphorylation events at CDK consensus sequences.
Measurement of the phosphorylation dynamics revealed a highly sensitive system poised to respond to CDK: turnover rates were on average two to three minutes (in a cell cycle of 2.5 hours), suggesting a huge sink of phosphatase activity driving futile cycles of dephosphorylation and phosphorylation — something that can be important in rapidly converting changes in CDK activity into stepwise threshold-dependent changes. Substrate turnover was the same throughout the cycle, implying that changing phosphatase activity plays no role in ensuring temporal order.
Over the course of the cell cycle, there were three different phosphorylation patterns: early, middle and late. Sixteen CDK substrates were first phosphorylated in S phase, their paucity perhaps illustrating the relative simplicity of this regulatory switch, and these then increased continuously in phosphorylation throughout the cell cycle; a further 12 sites had intermediate kinetics, and the vast majority were phosphorylated at G2/M.
Given that only one kinase, whose expression level increases progressively through the cell cycle, was involved, Swaffer and coworkers concluded that early in the cell cycle, low CDK activity results in phosphorylation of a few high affinity sites required for S phase, and these sites continue to be phosphorylated throughout the cell cycle. As CDK activity rises, medium and low affinity sites are successively phosphorylated. Crucially, and again contrary to the textbook view, there is no periodicity in phosphorylation: levels simply continue to accumulate throughout the cycle until the end of mitosis, when CDK is degraded and phosphorylation is reset to zero in preparation for the next cycle. Therefore, quantitative changes in CDK activity can bring about all the events of the cell cycle, and rising activity is the basic principle.
To confirm this hypothesis, the group developed a novel in vivo kinase assay. To dephosphorylate CDK substrates, cells were incubated at different stages of the cell cycle with the 1-NmPP1 inhibitor. Following inhibitor washout, the in vivo rate of CDK phosphorylation was measured, and was shown to increase by 50-100 fold during the cell cycle, illustrating the huge dynamic range of the kinase.
The difference in inherent CDK activity at early and late times can be mimicked by treatment with different concentrations of 1-NmPP1, and this approach was used to demonstrate that there were indeed high affinity early and low affinity late substrates in vivo: with low amounts of inhibitor, proteins which were normally rapidly phosphorylated late in the cell cycle ceased to be phosphorylated; conversely, inhibiting phosphorylation of proteins required early in the cycle required far more inhibitor.
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A quantitative model for cell cycle regulation by CDK
CDK is directly responsible for only a subset of the phosphorylation changes during the cell cycle, working with a set of other kinases to bring about progression through the different stages. Therefore, to understand how these and other kinases function together, Swaffer and coworkers next used phosphoproteomic analysis to produce a global description of how substrate phosphorylation changes during the cell cycle, working at a significantly higher temporal resolution than previous studies (Swaffer et al, 2018). Cells were released from G2 arrest and sampled at 20 timepoints across the cell cycle, to give a highly detailed picture of successive waves of phosphorylation and dephosphorylation. Given the evolutionary conservation of cell cycle regulation, this dataset is a significant community resource, useful for investigating cell cycle phosphoregulation across multiple eukaryotic systems, including higher vertebrates.
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Narrowing their focus to cell-cycle-regulated events, the group found that as cells passed through G1, CDK substrates were the last to be phosphorylated, acting as the final trigger to drive cells into S phase. This picture is markedly different to the situation later in the cycle. After cells complete S phase, CDK activity increases abruptly in late G2, phosphorylating hundreds of substrates to initiate mitotic onset, and it is absolutely required for activation of the three other mitotic kinases, Fin1, Plo1, and Ark1 (the fission yeast orthologues of NIMA-related, Polo-like and Aurora kinases respectively).
Analysis of the substrates of the mitotic kinases showed that each subset rose at different times during mitosis due to sequential, overlapping waves of phosphorylation by the kinases in the order: CDK, Fin1, Plo1 and Ark1. At exit from mitosis, dephosphorylation followed the same pattern. The time at which each kinase was activated was determined by the directness of its dependency on upstream CDK activity, with Ark1, the last kinase, independent of sustained CDK activity once cells had entered mitosis. These waves of activity overlay the microordering of cell cycle events imposed by the rising activity of CDK, producing a macroordering of the whole cell cycle.
The high quality of the mass spectrometry STP, and the expertise of colleagues working on protein phosphorylation and the cell cycle has created an excellent environment for our work to flourish.
Paul Nurse
In their most recent work, Nurse and colleagues have begun to dissect how the wide dynamic range of CDK activity across the cell cycle is achieved. Basu et al (2020) showed that a significant contribution to CDK’s in vivo activity is made by the kinase and its substrates being in the same place in the cell. Removal of the hydrophobic patch — the substrate docking region on CDK’s partner cyclin Cdc13 — resulted in a CDK which worked normally during S phase, but was unable to localise at later times to the spindle pole body (the fission yeast centromere equivalent), where a subset of CDK’s most refractory substrates are concentrated. CDK’s presence at the spindle pole body is essential for the cells to enter mitosis, and mutant CDK lacking the hydrophobic patch was unable to phosphorylate approximately half of all its mitotic substrates. Therefore, spatial organisation is a major factor in the increase in dynamic range of mitotic CDK: there is more kinase, but for it to work properly it has to be in the right place.
Given the high level of conservation of the cell cycle machinery, these new insights into cell cycle control are likely to have direct relevance in higher eukaryotes, providing a different view of how the cell cycle works. They also demonstrate that the increased complexity found in multicellular organisms, where there are multiple cyclin-dependent kinases and many more cyclins, is not important to the basic functioning of the cell cycle. Rather, the system in higher eukaryotes has likely evolved to meet the developmental and environmental challenges encountered by growing cells during the building and maintenance of an organism.
