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Identification of novel protein kinases required for meiosis

Final Report Summary - NPKM (Identification of novel protein kinases required for meiosis)

Meiosis is a specialised cell cycle that reduces the chromosome number by half to allow sexual reproduction. During this process, two rounds of chromosome segregation follow a single round of DNA replication. Complexity of meiotic chromosome segregation is pointing out the importance of the regulatory mechanisms that ensure the correct functions of all proteins involved in meiosis. Several lines of evidence suggest that proteins that are involved in meiosis are tightly regulated through their posttranslational modifications. In particular, protein phosphorylation has been demonstrated to be crucial in the proper regulation of these processes. However, for the vast majority of proteins that play important role in meiotic chromosome segegation the protein kinases responsible for their phosphorylation are not known.

Schizosaccharomyces pombe has at least 107 protein kinases from which 17 are known to be essential for cell growth. So far, most of meiotic studies focused on the functions of non-essential protein kinases. The studies of essential protein kinases were limited and relied mostly on conditional temperature-sensitive alleles. However, these alleles are often leaky, require analysis under non-physiological conditions (e.g. high temperature), or their inactivation requires a long period of time. Additionally, the elevated temperature has detrimental effects on meiosis, that result in chromosome missegregation.

To overwhelm the obstacles related to use of temperature-sensitive alleles of essential protein kinases, we took advantage of chemical genetic strategy and constructed functional conditional analog-sensitive alleles of all essential protein kinases of S. pombe. Mutating the gate-keeper residues in the ATP-binding pockets allowed us to inhibit the activity of essential protein kinases by small-molecule inhibitors that can bind only to the mutant protein kinases and not to any other wild type protein kinases.

Created conditional analog-sensitive protein kinase mutants allowed us systematically study all essential S. pombe protein kinases for their regulatory functions during meiosis. In our initial screen we analysed the meiotic role of essential protein kinases in strains carrying conditional analog-sensitive alleles of particular essential protein kinase with one copy of chromosome two labeled with GFP. To inactivate the kinase, we added inhibitor at the time when a large fraction of cells entered meiotic prophase. By scoring chromosome segregation we identified eight essential S. pombe protein kinases which function seems to be crucial for proper meiotic chromosome segregation.

In summary, identification of new essential protein kinases that are required for proper meiotic chromosome segregation, resulting from analysis of created conditional analog-sensitive alleles of essential protein kinases, was successful. Benefits of our results are really impressive. First, the created conditional analog-sensitive protein kinase mutants are freely available to other researchers. Availability of these analog-sensitive protein kinase alleles will help us to address various issues in different areas of cell biology and will allow other scientists to reveal so far hidden molecular mechanisms of these protein kinases. Secondly, a fuller understanding of the molecular mechanism of regulation of proteins that are involved in meiosis through their phosphorylation may help us to understand better how tightly these processes are regulated. From that point, our findings that several essential protein kinases are required for proper meiotic chromosome segregation is pushing us to continue in the detailed analysis of the role of selected essential protein kinases in recombination, mono-orientation of sister kinetochores and protection of centromeric cohesion during meiosis. Our better understanding of how is meiotic chromosome segregation regulated through phosphorylation may provide us with new opportunities for therapeutic methods.