Evolution of opposing regulatory interactions underlies the emergence of eukaryotic cell cycle checkpoints

Abstract

In eukaryotes the entry into mitosis is initiated by activation of cyclin-dependent kinases (CDKs), which in turn activate a large number of protein kinases to induce all mitotic processes. The general view is that kinases are active in mitosis and phosphatases turn them off in interphase. Kinases activate each other by cross- and self-phosphorylation, while phosphatases remove these phosphate groups to inactivate kinases. Crucial exceptions to this general rule are the interphase kinase Wee1 and the mitotic phosphatase Cdc25. Together they directly control CDK in an opposite way of the general rule of mitotic phosphorylation and interphase dephosphorylation. Here we investigate why this opposite system emerged and got fixed in almost all eukaryotes. Our results show that this reversed action of a kinase-phosphatase pair, Wee1 and Cdc25, on CDK is particularly suited to establish a stable G2 phase and to add checkpoints to the cell cycle. We show that all these regulators appeared together in LECA (Last Eukaryote Common Ancestor) and co-evolved in eukaryotes, suggesting that this twist in kinase-phosphatase regulation was a crucial step happening at the emergence of eukaryotes.

Figure 1

Similar dynamical behaviour of the mitotic entry and the mutual inhibition (MI) systems. (a) Wiring diagram of the Mutual Inhibition system (MI system). (b) Wiring diagram of the regulatory network of the G2/M transition. Colours of molecular species show their biological type: PP1, PP2 and Cdc25 are phosphatases (blue), and Gwl, Wee1 and Cdk1 are kinases (orange). Edges indicate catalytic reactions: ball-end activation; dash-end inhibition. Both networks are shown as influence networks (Supplementary Figure S1). Shadows of molecules in the G2M system point to their corresponding species in the MI system. (c) Time-course diagram of MI system. (d) Time-course diagram of the G2/M system. Diagrams show the active forms from each species. Active forms of Cdk1, Cdc25 and Gwl collapse into the ‘Mitosis’ trace of MI (brown), while active forms of PP2A, PP1 and Wee1 collapse into the ‘Interphase’ trace of MI (grey). See supplementary methods for details of the simulations. Graphics were obtained by R version 3.6 and ggplot2^,.

Figure 2

Dynamical analysis of General Kinase-Phosphatase (GKP system) and the mitotic entry network (G2/M system) driven by ATP. (a) Wiring diagram of the General Kinase-Phosphatase (GKP). Molecules grouped by biochemical function: blue scale phosphatases; orange scale kinase. Ball-end edges indicate activation; dash-end edges indicate inhibition. (b) Wiring diagram of the G2/M regulatory system (notations as on panel a). The atp label indicates the reactions which require these molecules. (c) Bifurcation diagram of the GKP system. It represents the steady-state concentration of the noted molecules at various ATP/ADP ratios. The system is bistable in the regime 0.77 < ATP/ADP < 1.53. (d) Bifurcation diagrams of the G2/M regulatory system representing the steady-state concentration of the molecules at various ATP/ADP ratios. Stable steady states labelled with thick, unstable steady states with thin dashed lines to allow the visualisation of overlapping curves. The system is bistable in the regime 0.71 < ATP/ADP < 7.77. The left-side panel shows the behaviour of Cdk1 and PP2A, while the right-side panel shows the rest of the species (Gwl, PP1, Wee1 and Cdc25) (e) Two-dimensional bifurcation diagram of the GKP and G2/M systems, showing how the bistable region changes as the rate of reactions (k₁) is changed from the baseline k1 = 1, which was used to plot panels c and d. The vertical dashed lines indicate the bistable region for the GKP and the G2/M systems (green and purple, respectively) at this k₁ level. See supplementary methods for details on the calculation of these curves. Graphics were obtained using R version 3.6 and ggplot2^,. For panel e, Oscill8 was used.

Figure 3

Arresting the entry into mitosis through checkpoint kinases. (a) Wiring diagram of the mitotic network controlled by the checkpoint kinase Chk. Blue palette indicates the phosphatases. Orange palette shows the kinases. Ball-end edges indicate activation; dash-end edges indicate inhibition. atp labels the edges that require a source of phosphate. (b-left) Bifurcation diagram of the steady-state concentration when the ATP/ADP ratio controls the system but Chk parameter is set at 0.5 (AU) (b-right) Bifurcation diagram of the steady-state concentration of the core cell cycle controllers with Chk as an external parameter and the ATP/ADP ratio is set to 8 (AU), which was enough to drive the cells into mitosis in the absence of Chk (Fig. 2). (c) Wiring diagram of the GKP system interacting with an external phosphatase through Pho2 and Kin1 (Chp-GKP system). Blue palette indicates the phosphatases. Orange palette labels the kinases. Ball-end edges indicate activation; dash-end edges indicate inhibition. atp labels the edges that require a source of phosphate. (d-left) Bifurcation diagram of the steady-state concentration when the ATP/ADP ratio drives the system and the Chp parameter is set at 0.5 (AU). (d-right) Bifurcation diagram of the steady-state concentration when Chp controls the system and the ATP/ADP ratio is set to 8 (AU). See supplementary methods for details of the simulations. R version 3.6 and ggplot2^, was used to create the plots.

Figure 4

Phylogenetic analyses of Wee1, Cdc25, Cdk1, Chk1 and Chk2. (a) Phylogenetic tree (424 species pruned to 100 species for clarity. See the complete phylogenetic tree in Supplementary Fig. S2) with the presence and absence of Chk2, Chk1, Cdk1, Cdc25 and Wee1. Wee1, Cdc25, Cdk1 and Chk2 were present in the last eukaryotic common ancestor (LECA, red arrow), and they were lost several times. Chk1 emerged in the common ancestor of Amorphea, based on ancestral state reconstruction (ASR) analysis. (b) Correlated evolution between Wee1, Cdc25, Cdk1, Chk1 and Chk2. Based on the Likelihood ratio test (LRT) results, Wee1 shows correlated evolution with Cdk1 and Chk1 significantly (≤ 0.05, LRT). Cdk1 shows correlated evolution with Cdc25 significantly (p ≤ 0.05, LRT) and Chk2 with high significance (p ≤ 0.001, LRT). (c) Patterns of evolutionary correlation between key kinases as well as proteins chosen as positive (Cks1) and negative controls (NDR). Wee1 shows correlated evolution with NDR (p ≤ 0.001, LRT), Cks1 shows correlated evolution with Cdc25, Cdk1, Chk2 (p ≤ 0.001, LRT), and with Chk1 (p ≤ 0.05, LRT).