Synthetic cell-cycle regulation identifies Mif2CENP-Cas a CDK phospho-target at the kinetochore

Abstract

AbstractProtein phosphorylation regulates multiple cellular processes including cell-cycle progression, which is driven by highly conserved cyclin-dependent kinases (CDKs). CDKs are controlled by the oscillating levels of activating cyclins and the activity peaks during mitosis to promote chromosome segregation. However, with some exceptions, we do not understand how the multitude of CDK-phosphorylated residues within the proteome drive cell-cycle progression nor which CDK phosphorylation events are necessary. To identify yeast proteins whose phospho-regulation is most critical for cell-cycle progression, we created a synthetic CDK complex and systematically recruited this to proteins involved in chromosome segregation using the Synthetic Physical Interactions (SPI) method. We found that targeted recruitment of synthetic CDK to the centromeric protein Mif2CENP-Cleads to enrichment of Mif2CENP-Cat centromeres and arrested cells in late mitosis. We then identified putative CDK consensus sites on Mif2CENP-Cwhich aid Mif2CENP-Clocalisation at centromeres and showed that CDK- dependent Mif2CENP-Cphosphorylation is important for its stable kinetochore localisation.SummaryTo identify cellular sites of functional cell cycle phospho-regulation we generated a synthetic cyclin-dependent kinase which can be recruited to any given GFP-tagged protein. Using this system with a set of proteins involved in chromosome segregation, we identified Mif2CENP-Cas a kinetochore target of CDK and show that CDK stabilises Mif2’s kinetochore localisation.