The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p

Abstract

CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk. CAK1 also has several CDC28-independent functions that are unique to meiosis. The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis. In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p. In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase. Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop. This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development. Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p. Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation. These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.

FIG. 1.

Cak1p is required for the timely destruction of Sic1p. Whole-cell lysates of CAK1 (JLY3) or cak1-Δ (JLY4) homozygous diploid cells in the CDC28-43244 background were prepared at the indicated times during sporulation and assayed by immunoblot analysis with Sic1p antiserum (α-Sic1). Vegetative growth is represented by the 0-h time point.

FIG. 2.

Depletion of Sic1p partially rescues the meiotic S-phase delay of cak1-Δ cells. Diploid strains containing a galactose-inducible SIC1 gene allowed depletion of Sic1p specifically during sporulation. (A) Progression through meiotic S phase was monitored by flow cytometry to detect cells with 2C and 4C DNA content as indicated (from left to right: JLY3, KSY112, JLY4, and KSY111). Sporulating cells were fixed at the indicated times postinduction, and DNA was stained with propidium iodide. (B) Completion of the meiotic divisions was monitored by fixing cells of the indicated genotype and staining the DNA with DAPI. Cells were viewed by microscopy under UV light and counted for completion of either MI or MII. All experiments were in the CDC28-43244 genetic background. (C) Protein was isolated from a cak1-Δ strain harboring the GAL1-SIC1 allele (KSY111) grown in inducing medium (galactose) or in noninducing medium (acetate) and at the indicated times after transfer to sporulation medium. Sic1p was monitored by immunoblot analysis.

FIG. 3.

Ime2p is unmodified in cak1-Δ strains. Protein was isolated from whole-cell lysates of sporulating cells at the indicated times from wild-type (W) (KSY137) or cak1-Δ (Δ) (KSY138) diploid cells in the CDC28-43244 genetic background. Ime2p-myc protein was assayed by immunoblot analysis with a polyclonal Ime2p antibody.

FIG. 4.

Cak1p is required for the catalytic activity of Ime2p. Epitope-tagged Ime2p was immunoprecipitated (IP) from sporulating CAK1 (KSY137) or cak1-Δ (KSY138) diploid cells in the CDC28-43244 background at the indicated times. Protein kinase activity was measured by incubation of Ime2p-myc with purified MBP-Ndt80p substrate and [γ-³²P]ATP. *P-Ime2 and *P-Ndt80 correspond to Ime2p autophosphorylation and phosphorylation of Ndt80p, respectively. Immunoblot analysis shows that comparable levels of Ime2p-myc are present in the reaction mixtures.

FIG. 5.

Cak1p is required for phosphorylation of Ime2p. The catalytically inactive ime2-K97R allele tagged with the myc epitope was introduced into the CDC28-43244 background in diploid cells either containing (KSY189) or lacking CAK1 (KSY190). Cells were sporulated for 2 h prior to lysis and partial purification of Ime2p by ion-exchange chromatography. Differences in the electrophoretic mobilities of Ime2p were detected by immunoblot analysis with an anti-myc antibody.

FIG. 6.

Ime2p T-loop residues T242 and Y244 are required for timely S phase. (A) The progression through meiotic S phase in diploid strains harboring IME2 T-loop point mutations (from left to right: KSY221, KSY222, and KSY220) was monitored by flow cytometry. Sporulating cells were fixed at the indicated times, and nuclear DNA was stained with propidium iodide to detect 2C and 4C populations. In the wild type (WT), 50% of cells complete S phase by around 3 h (see Fig. 2B for wild-type control). (B) Analysis of the electrophoretic mobilities of the IME2 T-loop mutants was conducted by comparing the epitope-tagged proteins in diploids containing CAK1 (W; from left to right: KSY137, KSY221, KSY222, and KSY220) and lacking CAK1(Δ; from left to right: KSY138, KSY223, KSY219, and KSY218). Whole-cell lysates from sporulating cells were prepared after 4 h and probed with an antibody against the myc epitope. Each pair represents the most clearly resolved image obtained, because the Cak1p-dependent modification of the single mutants resulted in a small, but reproducible, change in electrophoretic mobility. (C) The protein kinase activity of epitope-tagged Ime2p-myc mutants was assayed by mixing immunopurified Ime2p-myc with purified Ndt80p (upper panel). *P-Ime2 and *P-Ndt80 correspond to Ime2p autophosphorylation and phosphorylation of Ndt80p, respectively. The relative amounts of protein used in the reaction mixtures are shown in the lower panel by Western blot analysis of immunoprecipitated material.

FIG. 7.

Cdc28p-T169 phosphorylation levels do not change during sporulation. Whole-cell lysates taken from sporulating wild-type (WT) cells (LNY3) at the indicated times were probed with an antibody against Cdc28p (upper panel) and a phosphospecific antibody recognizing the T169 phosphorylated form of Cdc28p (lower panel). Strains containing and lacking CAK1 in the CDC28-43244 genetic background (JLY3 and JLY4, respectively) were used as controls.

FIG. 8.

Model for Cak1p's roles in meiosis. Cak1p functions through Ime2p to regulate S phase via Sic1p destruction and through Smk1p to regulate spore formation. Cak1p is also required to activate Cdc28p to initiate meiotic S phase and to execute the meiotic divisions. Not shown is that Cak1p activation of Ime2p also promotes middle gene induction, and thus M phase and spore formation, by inducing expression of CLBs and SMK1, respectively. Genes directly regulated by the transcriptional program of sporulation are underlined. X in the Ime2p and Smk1p pathways could be unique or shared and could represent Cak1p-activated kinases or noncatalytic regulatory components that promote the direct phosphorylation of Ime2p or Smk1p by Cak1p.