University of Dundee

EASTBIO: How do CDKs phosphorylate the right substrates at the right time?

Cell division is crucial for tissue renewal, wound repair, and the immune response. Cancer occurs when mechanisms that regulate cell division go wrong. The cell division cycle is controlled by the ordered phosphorylation of substrates by cyclin-dependent kinase (CDK) complexesIn yeast a single CDK can drive cell cycle progression, but in animal cells several different CDK complexes drive different stages of the cell cycle. How different CDK complexes target the right substrates is poorly understood. This PhD project will use a combination of biochemistry, genetics, structural biology, and cell biology to understand the mechanistic basis for temporal ordering of CDK substrate phosphorylation in human cells, and thus contribute to answering a key open question in the field.  


The Ly group has applied state of the art phosphoproteomics to dissect how non-catalytic subunits of CDKs contribute to substrate choice. The student will develop and apply novel kinase assays in which protein phosphorylation is induced in fixed and permeabilized human cells using recombinant CDK complexes. Global cellular phosphorylation levels are then measured using quantitative mass spectrometry. The kinase assay will allow the student to assess how substrate phosphorylation and substrate choice is affected by qualitative factors (i.e., the composition of the CDK complex) versus quantitative factors (i.e., concentration and kinetics). Our preliminary data on CDK1 surprisingly show that non-catalytic subunits of CDK1 can promote phosphorylation of non-proline directed phosphoacceptor sites, revealing a secondary consensus sequence specific to the non-catalytic subunit. These results suggest that contrary to textbook models, CDK1 can phosphorylate many non-proline directed sites in the human proteome. However, the function of these non-proline directed sites is unknownThe PhD project will explore the function of these non-canonical sites and investigate substrate phosphorylation by other CDKs, including CDK2 and CDK4/6.  


PhD research aims 


The interdisciplinary PhD project aims to dissect the biochemical regulation of CDK substrate phosphorylation in three parts:  


1. What is the role of the small Cks subunit in substrate phosphorylation by CDK2? 


2. What is the structural basis for CDK engagement with non-proline directed substrates? 


3. What is the function of non-proline directed phosphorylation by CDK in cells? 


The PhD student will have opportunities to learn techniques in biochemistry, molecular biology, cell biology, structural biology, and mass spectrometry-based proteomics. This multidisciplinary skillset will support professional development into a well-rounded researcher in the biological sciencesAdditionally, the student will be provided training in the analysis and visualization of proteomic datasets using the R scripting platform, which are skills that can be transferred to analysing large datasets.