Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2

Abstract

Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation. To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors. Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase. Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28. Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1. Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation. The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity. In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80. We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28.

Figure 1.

Cdc28 is required for meiotic DNA replication. (A) Chemical structure of 1-NM-PP1. (B) Flow cytometry of wild-type CDC28 (KBY316) and mutant cdc28-as1 (KBY442) cells sporulated with or without 5 μM 1-NM-PP1. DNA contents before (2C) and after (4C) DNA replication are indicated. (C) Proteins from cdc28-as1 cells. Protein extracts from KBY442 strain harvested during sporulation with or without 1-NM-PP1 were immunoblotted with antibodies against Ime2-myc (9E10), Sic1, and Ndt80. (D) Untagged Ime2 from sporulating wild-type (KBY259) cells was visualized with antibodies against Ime2. (E) Protein phosphatase treatment of Ime2-myc. Immunoprecipitates from IME2-myc (KBY316) cells harvested after 2 or 6 h of sporulation were divided and treated with (+) or without (-) λ protein phosphatase (phos.) before immunoblotting with the 9E10 antibody. Note that the phosphorylated forms of Ime2-myc are not resolved as well as those of untagged Ime2, because of the increased size of the tagged protein. (F) Protein phosphatase treatment of Ndt80-HA. Immunoprecipitate from NDT80-HA (KBY332) cells sporulated for 6 h was divided and treated with (+) or without (-) λ protein phosphatase (phos.) and phosphatase inhibitors (inh.) before immunoblotting with the 12CA5 antibody.

Figure 2.

Ime2 phosphorylates Ndt80 and exhibits a second peak of activity. (A) Activity assay for Ime2. Ime2-myc was immunoprecipitated from KBY316 cells harvested after 5 h of sporulation and incubated with [γ-³²P]ATP in the absence (none) or presence of additional proteins purified from E. coli: MBP alone or fused to full-length Ndt80 (MBP-Ndt80) or to a DNA-binding domain of Ndt80 (MBP-Ndt80DBD). Incorporation of radioactivity into Ime2-myc by autophosphorylation is indicated (*P-Ime2). Electrophoretic resolution of phosphorylated forms of Ndt80 was limited by the large MBP tag and the gel conditions. Protein size markers are indicated on the right. (B) Requirement of Ime2 catalytic activity for incorporation of radioactivity. IME2-myc (KBY316), ime2^K97R-myc (KBY450), and IME2 (KBY259) cells were processed for the kinase assay with MBP-Ndt80 substrate (top) or for immunoblotting with the 9E10 antibody (bottom). (C) Change in Ime2 activity during meiosis. Cells (KBY316) harvested after 0–8.5 h were assayed for kinase activity using MBP-Ndt80 substrate. Incorporation of radioactivity into MBP-Ndt80 (*P-Ndt80) and Ime2-myc (*P-Ime2) is indicated. (D) Timing of Ime2 activity, meiotic DNA replication, and meiotic divisions. The kinase activity quantified with a PhosphorImager from C is shown in arbitrary units. The percentages of cells containing more than 2C DNA content (DNA replication) or more than one DNA mass (meiotic divisions) are shown.

Figure 3.

The second peak of Ime2 activity occurs after pachytene. (A,B) Dependence of Ime2 kinase activity and protein on NDT80. Mutant ndt80Δ (KBY430) and wild-type (WT, KBY316) cells were harvested for the Ime2 kinase assay (A) and immunoblotting with 9E10 (B). (C–E) Requirement of Cdc28 for Ime2 activity and for Ime2 and Ndt80 proteins. Mutant cdc28-as1 (KBY442) and wild-type (WT, KBY316) strains were sporulated and treated with 0.5 μM 1-NM-PP1 at 2 h (C,D) or 5 μM 1-NM-PP1 at 0 h (E). Samples were examined by the Ime2 kinase assay (C,E) or immunoblotting to visualize Ime2-myc and Ndt80 (D). (F,G) Modification of untagged Ime2 in wild-type (KBY259), ndt80Δ (KBY427), and cdc28-as1 (KBY439) cells. The cdc28-as1 mutant was treated with 1-NM-PP1 as indicated. Multiple forms of Ime2 are indicated by a column of dots placed between 4and 7 h.

Figure 4.

The ime2-as1 mutation confers sensitivity to 1-NA-PP1. (A) Chemical structures of 1-NA-PP1 and Bn-PP1. (B–D) Wild-type (WT, KBY316) and mutant ime2-as1 (KBY518) cells sporulated in the presence (+) or absence (-) of 20 μM 1-NA-PP1 were harvested for flow cytometry (B), DAPI staining (C), and immunoblotting (D). (C) Meiotic divisions are shown as the percentage of cells with more than one DAPI-staining body. (D) Protein extracts were immunoblotted with antibodies against Ime2-myc (9E10), Sic1, and Ndt80.

Figure 5.

Ime2 is required for the meiotic nuclear divisions. (A) Meiotic divisions in ime2-as1 (KBY518) cells treated with 20 μM 1-NA-PP1 at 2, 4, or 6 h of sporulation. The percentage of cells in each sample with more than one DAPI-staining body is shown. (B–D) Sporulating ime2-as1 (KBY518) cells were treated with (+) or without (-) 20 μM 1-NA-PP1 at 5.25 h and harvested at 4–10.25 h of sporulation for analysis. (B) DAPI-stained cells were scored for the occurrence of meiosis I (MI, 2 DAPI bodies) and meiosis II (MII, 4 DAPI bodies). (C) Protein extracts were immunoblotted with antibodies against Ime2-as1-myc (9E10) and Ndt80. (D) Northern blots were probed to visualize IME2; NDT80; the middle sporulation genes CLB1, CLB4, SMK1, SPS1, SPS4, and SPR3; and the loading control PFY1; then quantitated. Normalized transcript levels are shown. Before 1-NA-PP1 was added, transcript levels of all genes (except PFY1) had increased at least 30-fold relative to the 0-h time point. (E) Sporulating ime2-as1 (KBY518) cells were incubated with 60 μg/mL benomyl at 3.5 h and treated with (+) or without (-) 10 μM Bn-PP1 at 7 h. Cells were washed free of benomyl at 7.5 h and resuspended in medium with or without Bn-PP1. DAPI-stained cells were scored as in B.

Figure 6.

Increased expression of NDT80 partially rescues the meiotic division defect of Ime2-deficient cells. (A–E) Transcription from P_GAL-NDT80 was induced at 3 h by addition of 0.5 μM β-estradiol to sporulating NDT80 ime2-as1 (KBY518) and P_GAL-NDT80 GAL4-ER ime2-as1 (KBY649) cells. At 4 h, cultures were split and treated with 10 μM Bn-PP1 (+) or not (-). (A) Amount of NDT80 transcript shown in arbitrary units (a.u.). (B) Immunoblots showing production and phosphorylation of Ndt80 protein. Ime2 is shown as a loading control. (C) Graph of meiotic divisions in cells with wild-type or inducible NDT80. The percentage of cells in each sample with more than one DAPI-staining body is shown. (D) Impaired meiotic divisions after inhibition of Ime2-as1. DAPI-stained cells were scored for the occurrence of meiosis I (MI, 2 DAPI bodies) and meiosis II (MII, 4DAPI bodies). (E) Transcript levels of representative middle genes. The CLB1 profile shown is similar to that of CLB4, CLB5, SMK1, and SPO77. The SPS1 profile is similar to that of SPR3 and SPS4. (F) Phosphorylation of Ndt80 in an ime2Δ strain with inducible NDT80. IME2 (KBY373) and ime2Δ (KBY387) strains containing inducible NDT80 (P_GAL-NDT80 GAL4-ER) were sporulated for 6 h in the absence (-) or presence (+) of 5 μM β-estradiol and harvested for immunoblotting with Ndt80 antibodies. A heterozygous control strain (C, KBY375) contained both wild-type and inducible NDT80 genes. (G) The immunoblot shows phosphorylation of Ndt80 in wild-type (KBY316) and ime2Δ sum1Δ mutant (KBY619) cells.

Figure 7.

Pathway of meiotic progression. See text for details. Cdc28 and Ime2 are required for both early (DNA replication) and late (nuclear divisions) processes in meiosis. (A) Early: An early form of Ime2 (Ime2_E) antagonizes Sic1 and promotes Ndt80 activity at both transcriptional and posttranslational levels. An early activity of Cdc28 (Cdc28_E) is essential for meiotic DNA replication. (B) Middle: Ndt80 turns on transcription of its own gene in an autoactivation loop that helps boost transcription of the middle sporulation genes. (C) Late: Late activities of Cdc28_L and Ime2_L are necessary for the meiotic divisions. Ndt80 and Cdc28_L stimulate activity, accumulation, and hyperphosphorylation of Ime2_L. In turn, Ime2_L is needed to maintain high levels and phosphorylation of Ndt80, to promote full transcription of middle genes, and thus to ensure adequate Cdc28_L/Clb kinase activity. Ime2_L may also play an NDT80-independent role in the meiotic divisions.