Cdc28 activates exit from mitosis in budding yeast

Abstract

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.

Figure 1

CDC28-VF delays in exit from mitosis. A, Wild-type (ADR1859) and CDC28-VF (ADR1857) were grown overnight at 23°C in YPD to mid log phase, were arrested in G1 with alpha factor (1 μg/ml), and at t = 0 the cells were released from the G1 arrest. At t = 90, alpha factor (1.5 μg/ml) was added back to the cultures to rearrest the cells in the next G1. Left, Samples were taken at the indicated times and processed for Western blots and histone H1 kinase assays. Right, Sister chromatid separation was scored by counting the number of fluorescent spots (one or two) of GFP-lacI bound to 256 tandem repeats of lacO integrated at the TRP1 locus. B, Wild-type (ADR548) and CDC28-VF (JM477) were grown overnight at 23°C in YEP + 2% glucose to log phase, were arrested in mitosis with nocodazole (10 μg/ml) for 3 h, and at t = 0 the cells were released from the arrest into fresh medium containing alpha factor (10 μg/ml). Samples were taken at the indicated times and processed for Western blots and histone H1 kinase assays. Sister chromatid separation was scored by counting the number of fluorescent spots (one or two) of GFP-lacI bound to 256 tandem repeats of lacO integrated at the LEU2 locus.

Figure 2

CDC28-VF cannot overcome a spindle checkpoint-dependent arrest. A, Serial dilution assay. All strains contain pGAL-MPS1. Wild-type (KH153), CDC28-VF (KH181), mad3Δ (ADR1248), and CDC28-VF mad3Δ (KH183) were grown overnight in YEP + 2% glucose to log phase and adjusted to A₆₀₀ 0.35. Twofold serial dilutions of each strain were prepared in a multiwell dish and then spotted onto YEP + 2% glucose (left) or YEP + 2% galactose (right). The plates were incubated at 30°C for 2 d. B, Microcolony assay. The same strains as in A were grown overnight at 30°C in YEP + 2% raffinose to log phase and at t = 0, unbudded cells were picked out onto YEP + 2% galactose plates. As each cell divided, the number of cells in each microcolony were counted at the indicated times. Large-budded cells were counted as two cells and the original cells that did not bud were not included in the analysis. Each strain has been tested at least three times, following 20–30 cells of each strain in each experiment. In the experiment shown, 30 cells of each strain were followed.

Figure 3

Changes in inhibitory phosphorylation on Cdc28 do not affect sensitivity to the spindle checkpoint. A, Serial dilutions of strains with varying amounts of inhibitory phosphorylation on Cdc28. All strains contain pGAL-MPS1. Wild-type (KH153), CDC28-VF (KH181), CDC28-V (ADR1105), CDC28-F (KH204), swe1Δ (KH207), swe1Δ CDC28-V (ADR1100), CDC28-AF (ADR1506), and mih1Δ (ADR1378) were grown to saturation for 2 d in YEP + 2% glucose at 30°C, diluted tenfold, and fourfold serial dilutions were prepared in a multiwell dish and spotted onto YEP + 2% glucose (left) or YEP + 2% galactose (right). The plates were incubated at 30°C for 2 d. B, Phosphotyrosine blot. Wild-type (ADR477), CDC28-VF (ADR509), cdc55Δ (JM445), swe1Δ (ADR684), and mih1Δ (ADR1314) were grown overnight in YEP + 2% glucose at 23°C to log phase. The cells were harvested, lysed, and Cdc28-HA was immunoprecipitated with the 12CA5 antibody. The immunoprecipitates were run out on a polyacrylamide gel and probed either with a phosphotyrosine antibody (top) or with 12CA5 (bottom).

Figure 4

Mutants with defects in mitotic Cdc28 activity resemble CDC28-VF. A, Defects in mitotic Cdc28 activity are sensitive to spindle checkpoint-dependent arrest. All strains contain pGAL-MPS1. Wild-type (KH153), CDC28-VF (KH181), cdc28-1N (ADR1899), cdc28-4 (ADR1901), clb2Δ (ADR1606), and cks1-38 (ADR1903) were grown to saturation for 2 d in YEP + 2% glucose at 23°C, diluted tenfold, and fourfold serial dilutions were prepared in a multiwell dish and spotted onto YEP + 2% glucose (left) or YEP + 2% galactose (right). The plates were incubated at 23°C for 2.5 d. B, The specific activity of Cdc28-VF is lower than Cdc28. Wild-type (ADR477) and CDC28-VF (ADR509) were grown overnight in YEP + 2% glucose at 23°C to log phase and arrested in mitosis with nocodazole (10 μg/ml) for 3 h. The cells were then harvested, lysed, and Clb2/Cdc28 and Clb3/Cdc28 complexes were immunoprecipitated, and their histone H1 kinase activity was measured. The Western blot (bottom) shows that equal amounts of Cdc28 are precipitated in the two strains, although the kinase activity (top) of Cdc28-VF is reduced relative to wild-type. The activity of wild-type Cdc28 is reported as 100% for both the anti-Clb2 and anti-Clb3 immunoprecipitates. C, CDC28-F88G behaves like CDC28-VF. All strains contain pGAL-MPS1. Wild-type (KH153), CDC28-VF (KH181), and CDC28-F88G (ADR2034) were grown to saturation for 2 d in YEP + 2% glucose at 30°C, diluted tenfold, and fourfold serial dilutions were prepared in a multiwell dish and spotted onto YEP + 2% glucose (left) or YEP + 2% galactose (right). The plates were incubated at 30°C for 2 d. D, 2μ-CDC28 suppresses CDC28-VF. All strains contain pGAL-MPS1. Wild-type (KH153) or CDC28-VF (KH181) containing either 2μ-CDC28 or an empty 2μ vector were grown to saturation for 2 d in CSM-trp + 2% glucose at 30°C, diluted fivefold, and fourfold serial dilutions were prepared in a multiwell dish and were spotted onto CSM-trp + 2% glucose (left) or CSM-trp + 2% galactose (right). The plates were incubated at 30°C for 2 d.

Figure 5

cdc23-1 GAL-CDC28-VF arrest in mitosis at the permissive temperature of 23°C. A, cdc23-1 GAL-CDC28 (ADR1685) and cdc23-1 GAL-CDC28-VF (ADR1687) were grown overnight at 23°C in YEP + 2% raffinose to log phase, arrested in G1 with alpha factor (1 μg/ml) for 3.5 h, and at t = 0 released from the G1 arrest into fresh YEP + 2% galactose. After cells had budded (t = 2), alpha factor (1.5 g/ml) was added back to the cultures to rearrest the cells in the next G1. Samples were taken at the indicated times and processed for Western blots and histone H1 kinase assays. B, Sister chromatid separation was scored by counting the number of fluorescent spots (one or two) of GFP-lacI bound to 256 tandem repeats of lacO integrated at the TRP1 locus.

Figure 6

CDC28-VF has normal G1 APC activity. Wild-type (ADR1389) and CDC28-VF (ADR1252) were grown overnight at 30°C in YEP + 2% glucose to log phase and arrested in G1 with alpha factor (1 μg/ml) for 3 h. The cells were harvested, lysed, and the APC was immunoprecipitated with anti-Cdc26 antibodies. The in vitro ubiquitination activity of the immunoprecipitates was measured in the absence or presence of added Hct1 protein (left). The substrate for the in vitro ubiquitination is an iodinated NH₂-terminal fragment of sea urchin cyclin B (CycB). Western blotting of the immunoprecipitates shows that equal amounts of Cdc16, Cdc23, and Cdc27 are present in the APC isolated from wild-type and CDC28-VF cells (right).

Figure 7

CDC28-VF cells have a defective Cdc20-dependent APC. A, Destruction of Pds1 in anaphase depends on CDC20. Wild-type (ADR1870), cdc20-3 (ADR1783), and hct1Δ (ADR1786) were grown overnight at 23°C in YEP + 2% raffinose to log phase. The three strains contain an epitope-tagged Pds1 (PDS1-myc18), can overexpress a truncated form of Clb2 (pGAL-CLB2-Δ176), which will arrest cells in anaphase, and express a GFP-tagged alpha tubulin (pHIS3-GFP-TUB1), which allows the length of the spindle to be easily assessed by microscopy. Wild-type and cdc20-3 were arrested in G1 by alpha factor (1 μg/ml) for 3.5 h, released from the G1 arrest into YEP + 2% galactose, and at t = 0, when >90% of the cells had reached anaphase (after 5 h, as judged by spindle length), the cultures were shifted to 37°C. hct1Δ, which is resistant to alpha factor, was shifted from YEP + 2% raffinose directly to YEP + 2% galactose. Samples were taken at the indicated times and processed for Western blots. B, Pds1 is stable in anaphase in CDC28-VF and clb2Δ. cdc15-2 GAL-PDS1-HA (ADR1743), cdc15-2 CDC28-VF GAL-PDS1-HA (ADR1736), and cdc15-2 clb2Δ GAL-PDS1-HA (ADR1774) were grown overnight at 23°C in YEP + 2% raffinose to log phase and shifted to 37°C to arrest the cells in anaphase (raf). When >90% of the cells had reached anaphase (after 4 h, as judged by nuclear division, which was scored by DAPI staining), Pds1-HA expression was induced for 1 h by the addition of galactose (to 2%), and at t = 0 its expression was terminated by the addition of glucose (to 2%). Samples were taken at the indicated times and processed for Western blots. Cdc28-HA is shown as a loading control. C, The Hct1-dependent APC regulates Pds1 stability in G1. CDC28-HA GAL-PDS1-HA (ADR1968), CDC28-VF-HA GAL-PDS1-HA (ADR1959), and cdc20-3 GAL-PDS1-HA (ADR1921) were grown overnight at 23°C in YEP + 2% raffinose to log phase and arrested in G1 with alpha factor (1 μg/ml) for 3 h at 23°C, and then shifted to 37°C for an additional 1 h (raf). cdc28-13 GAL-PDS1-HA (ADR1925) and cdc28-13 hct1Δ GAL-PDS1-HA (ADR1928) were grown overnight at 23°C in YEP + 2% raffinose to log phase and arrested in G1 by shifting the cultures to 37°C for 3.5 h. Pds1-HA expression was induced by addition of galactose (to 2%) for 2 h, and at t = 0 its expression was terminated by the addition of glucose (to 2%). Samples were taken at the indicated times and processed for Western blots. Sic1 is shown as a loading control and as evidence that all cells remain arrested in G1.

Figure 8

Cdc20 binding to the APC is impaired in CDC28-VF. cdc15-2 (ADR1790) and cdc15-2 CDC28-VF (ADR1793) were grown overnight in YEP + 2% glucose at 23°C to log phase. Both strains contain an epitope-tagged Cdc20 (CDC20-myc12). The cultures were shifted to 37°C and after >85% of the cells were arrested in anaphase (4 h, as judged by nuclear division, which was scored by DAPI staining), the cells were harvested, lysed, and the APC immunoprecipitated with anti-Cdc26 antibodies. The amount of Cdc20-myc12 bound to the APC was determined by Western blotting the immunoprecipitates with the 9E10 antibody. Equal amounts of APC were precipitated with the anti-Cdc26 antibodies as judged by Cdc23 levels (left) and equal amounts of cell lysate were used in the immunoprecipitation as judged by Cdc20-myc12 and Cdc28-HA levels (right, cell lysate).