FellowIng., PhD. Lubos Cipak
Project NameIdentification of substrates of essential protein kinases using "shokat" mutants
Host organisationBiomedical Research Center
Duration of the project04.01.2015 - 31.03.2018

Abstract
Genomic instability is defined as a process prone to genomic changes or an increased propensity for genomic alterations. The ultimate goal of cell division for most cells is to accurately duplicate the genome and then evenly divide the duplicated genome into the daughter cells or into the germ cells. Alterations or abnormally high-frequency of errors during processes involved in cell division, such as splicing, sister chromatid cohesion, kinetochore assembly and attachment, chromosome segregation or cytokinesis would cause missegregation of chromatids and chromosomal instability. The complexity of cell division is pointing out that this process is controlled by multi-level regulatory network. Several lines of evidence demonstrated that posttranslational modifications of proteins by phosphorylation play important role in regulation of protein functions. However, we still have little clue which protein kinases regulate proteins involved in processes of cell division. So, identification of relevant protein kinases and characterization of biological significance of their activities is highly desirable. Proposed project is aimed to identify direct targets of essential protein kinases and characterize functional relevance of phosphorylation of target proteins for regulation of splicing and fidelity of chromosome segregation by combining the power of analog-sensitive alleles of essential protein kinases with affinity purification and mass spectrometry approaches. The project will provide a new framework for our understanding of how are splicing and chromosome segregation regulated by phosphorylation and will lay the groundwork for future studies. Since the basic cell cycle machinery is highly conserved from yeast to man, it is likely that processes governing in yeast will guide the way for studies of these processes in higher eukaryotes, including humans.

Project Summary with Interim Results

Genomic instability is defined as a process prone to genomic changes or an increased propensity for genomic alterations. Alterations or abnormally high-frequency of errors during processes involved in cell division, such as pre-mRNA splicing, DNA repair or chromosome segregation would cause genome instability. The complexity of cell division is pointing out that these processes are controlled by multi-level regulatory network. Protein kinases are key regulators that direct the activity, localization and overall function of many proteins, and serve to orchestrate the activity of almost all cellular processes.

Proposed project is aimed to identify direct targets of essential protein kinases and characterize functional relevance of phosphorylation of target proteins for regulation of splicing and fidelity of chromosome segregation. So far, we successfully performed the experiments leading to identification of the substrates of protein kinases in fission yeast S. pombe and obtained tangible evidence that activities of protein kinases might be linked to pre-mRNA processing and cohesin functions. Mass spectrometry analysis of isolated protein kinase complexes revealed that proteins forming these complexes are phosphorylated on several serine, threonine, or tyrosine residues. Undergoing detailed phenotypical/genetic analysis of created phosphomutants will increase our understanding of how are splicing and chromosome segregation regulated and will lay the groundwork for future studies.