Roles of the mitotic inhibitors Wee1 and Mik1 in the G(2) DNA damage and replication checkpoints

Abstract

The G(2) DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases. However, the in vivo role of these kinases in checkpoint regulation has been unclear. We show that, in the fission yeast Schizosaccharomyces pombe, Mik1 is a target of both checkpoints and that the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints. Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance. In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint.

FIG. 1

Constitutive overexpression of Pyp3 rescues cdc25Δ. (A) Wild-type (PR109), nmt1:pyp3⁺ cdc25Δ (NR2613), and nmt1:pyp3⁺ cdc25Δ wee1-3x (NR2640) cells were grown in synthetic media lacking thiamine to induce high levels of expression of Pyp3 from the nmt1 promoter. The inset is the average length at septation for at least 50 cells, plus or minus the standard deviation. (B and C) wee1-50ts mik1Δ (PR754) and nmt1:pyp3⁺ cdc25Δ wee1-50ts mik1-s14ts (NR2644) cells were grown at 25°C, elutriated to collect a synchronous population of cells, and shifted to 35°C. The irradiated cultures were continuously irradiated with gamma radiation at 1 Gy/min from 30 min before the shift. The HU-blocked cultures were grown in 10 mM HU for 60 min before elutriation, so that the elutriated cells would be blocked in S phase. Half of the culture was then released from the HU block and allowed to complete replication before the shift to 35°C. Cell cycle progression was monitored microscopically. For clarity, the data from the HU-released cultures is not shown, but those cultures divided with kinetics similar to that of the untreated cultures. The cell cycle kinetic data shown here and in the other figures are representative of at least three similar experiments.

FIG. 2

The phosphorylation of Cdc2 by Mik1, but not Wee1, is up-regulated by the DNA damage checkpoint. (A through D) Wild-type (PR109), nmt1:pyp3⁺ cdc25Δ (NR2613), nmt1:pyp3⁺ cdc25Δ wee1-50ts (NR2630), and nmt1:pyp3⁺ cdc25Δ mik1Δ (NR2634) cells were elutriated and irradiated with 100 Gy of gamma radiation from approximately 2 h before the midpoint of septation, as indicated by the bracket (irradiated), or mock irradiated (unirradiated). Cell cycle progression was monitored microscopically. (E) nmt1:pyp3⁺ cdc25Δ mik1⁺:13Myc (NR2650) cells were grown to logarithmic phase and irradiated with 100 Gy of gamma radiation or incubated for 2 h with 10 mM HU. At indicated times, samples were taken and cleared whole-cell extracts were prepared. The extracts were analyzed by Western blotting with monoclonal anti-Myc antibodies (9E10; Covance). The slightly greater signal in the asynchronous sample compared with that in the irradiated samples is due to the small percentage of S-phase cells in the asynchronous sample, which produce higher levels of Mik1. (F) chk1⁺:9Myc (BF2521), rad3Δ chk1⁺:9Myc (NR2648), and nmt1:pyp3⁺ cdc25Δ chk1⁺:9Myc (NR2646) cells were grown to logarithmic phase, irradiated with 100 Gy of gamma radiation, and analyzed as described for panel E.

FIG. 3

Mik1 is regulated by the DNA damage checkpoint. (A and B) wee1-50ts cdc25-22ts (KS1362), wee1-50ts cdc25-22ts mik1Δ (PR1928), and wee1-50ts mik1Δ (PR754) cells were elutriated and shifted to 35°C at 20 min. Cells were irradiated with 100 Gy of gamma radiation from 30 to 60 min, irradiated with 50 J/m² at 60 min, or treated with 5 mU of bleomycin from 60 min. (C) Wild-type (PR109), wee1-50ts cdc25-22ts (KS1362), and wee1-50ts cdc25-22ts mik1Δ (PR1928) cells were grown at 25°C, shifted to 35°C for 40 min to inactivate wee1-50ts and cdc25-22ts, and irradiated with 100 J/m² or treated with 2.5 U/of bleomycin per ml. Septation was monitored microscopically and normalized to the zero time point. The septation index of wee1-50ts cdc25-22ts mik1Δ cultures rises as the cells enter mitotic catastrophe (26). (D) wee1-50ts cdc25-22ts mik1Δ (PR1928) and wee1-50ts mik1Δ (PR754) cells were elutriated, irradiated with 100 Gy of gamma radiation from 0 to 30 min, and shifted to 35°C at 30 min.

FIG. 4

Phosphorylation of Cdc2 by Mik1, but not Wee1, is up-regulated by the DNA replication checkpoint. (A through D) Wild-type (PR109), nmt1:pyp3⁺ cdc25Δ (NR2613), nmt1:pyp3⁺ cdc25Δ wee1-50ts (NR2630), and nmt1:pyp3⁺ cdc25Δ mik1-s14ts (NR2657) cells were grown for 60 to 90 minutes in 10 mM HU and elutriated to produce a synchronous culture arrested in S phase. Half of the culture was left in HU (HU blocked), while HU was washed out of the other half (HU released), which quickly went through DNA replication and then divided with kinetics similar to that of untreated cultures. Cell cycle progression was monitored microscopically. (E) Wild-type (PR109), cds1Δ (NB2117), rad3Δ (NR1826), nmt1:pyp3⁺ cdc25Δ (NR2613), nmt1:pyp3⁺ cdc25Δ wee1-50ts (NR2630), and nmt1:pyp3⁺ cdc25Δ mik1Δ (NR2634) cells were grown to logarithmic phase and incubated for 4 h with 10 mM HU. Cds1 activity was assayed by its ability to bind and phosphorylate the amino terminus of Wee1 (6).

FIG. 5

Model for the regulation of mitosis by the G₂ DNA replication checkpoints. Initiation of mitosis is controlled by the tyrosine dephosphorylation of Cdc2. The timing of this dephosphorylation is regulated by a balance between the activities of the Wee1 and Mik1 tyrosine kinases on one hand and the activity of the Cdc25 phosphatases on the other. In order to delay mitosis, the G₂ DNA damage and DNA replication checkpoints, acting through their effector kinases, Chk1 and Cds1, regulate both Cdc25 and Mik1. Chk1 inhibits the dephosphorylation of Cdc2 by phosphorylating and inhibiting Cdc25. In addition, Chk1 up-regulates Mik1 in two separate ways. It does so as an immediate response to the checkpoint, and this up-regulation is important for checkpoint establishment. It also leads to the accumulation of Mik1 protein during prolonged checkpoints, and this accumulation may be important for checkpoint maintenance. The role of Mik1 regulation in the establishment of the DNA damage checkpoint is minor compared with that of Cdc25, as indicated by the dashed arrow. In a manner similar to that of Chk1, Cds1 inhibits the dephosphorylation of Cdc2 by phosphorylating and inhibiting Cdc25. Cds1 also up-regulates Mik1, but to a much greater extent than Chk1. The up-regulation by Cds1 correlates with, and is presumably due to, high levels of Mik1 accumulation in DNA replication-arrested cells.