Ime2p and Cdc28p: co-pilots driving meiotic development

Meiosis can be considered an elaboration of the cell division cycle in the sense that meiosis combines cell-cycle processes with programs specific to meiosis. Each phase of the cell division cycle is driven forward by cell-cycle kinases (Cdk) and coordinated with other phases of the cycle through checkpoint functions. Meiotic differentiation is also controlled by these two types of regulation; however, recent study in the budding yeast S. cerevisiae indicates that progression of meiosis is also controlled by a master regulator specific to meiosis, namely the Ime2p kinase. Below, I describe the overlapping roles of Ime2p and Cdk during meiosis in yeast and speculate on how these two kinases cooperate to drive the progression of meiosis.     

Distinct activities of the related protein kinases Cdk1 and Ime2

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin-CDK complexes Cln1-Cdk1 and Cln2-Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin-CDK complexes are not involved. It has been proposed that the meiosis-specific protein kinase Ime2 functionally replaces the G1 cyclin-CDK complexes to promote Sic1 destruction. To investigate this possibility, we compared Cln2-Cdk1 and Ime2 protein kinase activities in vitro. Both enzyme preparations were capable of catalyzing phosphorylation of a GST-Sic1 fusion protein, but the phosphoisomers generated by the two activities had significantly different electrophoretic mobilities. Furthermore, mutation of consensus CDK phosphorylation sites in Sic1 affected Cln2-Cdk1- but not Ime2-dependent phosphorylation. Phosphoamino acid analysis and phosphopeptide mapping provided additional evidence that Cln2-Cdk1 and Ime2 targeted different residues within Sic1. Examination of other substrates both in vitro and in vivo also revealed differing specificities. These results indicate that Ime2 does not simply replace G1 cyclin-CDK complexes in promoting Sic1 degradation during meiosis.     

Ectopic expression of human Cdk2 and its yeast homolog,Ime2, is deleterious to Saccharomyces cerevisiae

Entry into and precise progression through the cell cycle depends on the sequential expression and activation of cyclin dependent kinases (CDK). In accord, CDK dysregulation is a hallmark of many cancers. The function of Cdk2 is still an enigma as in vitro studies revealed that it is required for S phase-entry, whereas in vivo studies showed that Cdk2 is not an essential gene. Moreover, unlike other Cdks, or its cyclin E regulator, Cdk2-overexpressing tumors were reported only in one type of tumor. In this report we used budding yeast as a tool to explore Cdk2 function. We showed that hCdk2 promoted S phase in cells carrying a temperature-sensitive mutation in yCDK1, albeit, only when expressed at low or moderate levels. Overexpression of hCdk2 resulted in a defect in the G1 to S transition and a reduction in viability. The same phenotypes were observed in cells overexpressing its yeast functional homolog, Ime2, which is a meiosis-specific CDK-like kinase. A genetic interaction with the DNA damage checkpoint was demonstrated by showing an increased toxicity of hCdk2 and Ime2 in RAD53-deleted cells, and delayed Rad53 activation in response to MMS treatment in cells overexpressing hCdk2 or Ime2.     

The Ime2 protein kinase family in fungi: more duties than just meiosis

Ime2 of the budding yeast Saccharomyces cerevisiae belongs to a family of conserved protein kinases displaying sequence similarities to both cyclin-dependent kinases and mitogen-activated protein kinases. Ime2 has a pivotal role for meiosis and sporulation. The involvement of this protein kinase in the regulation of various key events in meiosis, such as the initiation of DNA replication, the expression of meiosis-specific genes and the passage through the two consecutive rounds of nuclear divisions has been characterized in detail. More than 20 years after the identification of the IME2 gene, a recent report has provided the first evidence for a function of this gene outside of meiosis, which is the regulation of pseudohyphal growth. In the last few years, Ime2-related protein kinases from various fungal species were studied. Remarkably, these homologues are not generally required for meiosis, but instead have other specific tasks. In filamentous ascomycete species, Ime2 homologues are involved in the inhibition of fruiting body formation in response to environmental signals. In the pathogenic basidiomycetes Ustilago maydis and Cryptococcus neoformans, members of this kinase family apparently have primary roles in regulating mating. Thus, Ime2-related kinases exhibit an amazing variety in controlling sexual developmental programs in fungi.     

Cdc28 and Ime2 possess redundant functions in promoting entry into premeiotic DNA replication in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2Delta diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2Delta diploids depends on a functional Cdc28. Ime2Delta cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.     

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2ΔC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.     

An InCytes from MBC Selection: Regulation of Cell Polarity through Phosphorylation of Bni4 by Pho85 G1 Cyclin-dependent Kinases in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1,2-Cdc28 and Pcl1,2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, the redundancy of Cdks and cyclins means that identification of relevant Cdk substrates remains a significant challenge. We used array-based genetic screens (synthetic genetic array or SGA analysis) to dissect redundant pathways associated with G1 cyclins and identified Bni4 as a substrate of the Pcl1- and Pcl2-Pho85 kinases. BNI4 encodes an adaptor protein that targets several proteins to the bud neck. Deletion of BNI4 results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 is toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. Phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to the bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1Δpcl2Δ mutant cells. Our data suggest that misregulated Bni4 may bind in an uncontrolled manner to an essential component that resides at the bud neck, causing catastrophic morphogenesis defects.     

Therapeutic S and G2 checkpoint override causes centromere fragmentation in mitosis

In budding yeast, the meiosis-specific protein kinase Ime2 is required for normal meiotic progression.Current evidence suggests that Ime2 is functionally related to Cdc28, the major cyclin-dependent kinase in yeastthat is essential for both cell cycle and meiosis. We have previously reported that a natural target of Ime2 activityis replication protein A (RPA), the cellular single-stranded DNA-binding protein that performs critical functionsduring DNA replication, repair, and recombination. Ime2-dependent RPA phosphorylation first occursearly in meiosis and targets the middle subunit of the RPA heterotrimeric complex (Rfa2). We now demonstratethat Rfa2 serine 27 (S27) is required for Ime2-dependent Rfa2 phosphorylation in vivo. S27 is also required forRfa2 phosphorylation in vitro catalyzed by immunoprecipitated Ime2. In addition, Ime2 mediates in vitro phosphorylationof a short peptide containing Rfa2 amino acids 23 through 29, thereby providing evidence that S27itself is the phosphoacceptor. Phosphorylation site mapping supports this conclusion, as mass spectrometryanalysis has revealed that at least three residues within Rfa2 amino acids 2 through 35 become phosphorylatedspecifically during meiosis. Although S27 is embedded in a motif that is recognized by several protein kinases,this sequence is not a typical target of cyclin-dependent kinases. Therefore, the mechanism underlying Ime2substrate recognition could differ from that of Cdc28.     

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.