Supervisors : Dr Tony Ly and Professor Julian Blow, Division of Gene Regulation and Expression.
Cell division is crucial for tissue renewal, wound repair, and the immune response. Cancer occurs when mechanisms that regulate cell division go wrong. Cyclin-dependent kinases (CDKs) are master drivers of the cell division cycle, by coordinating the timing and progression of DNA replication and mitosis. CDKs phosphorylate cellular proteins to modulate their functions in a temporally scheduled manner. Temporal phosphorylation fidelity is crucial: premature CDK activation causes genome instability, which contributes to human disease. But how CDKs phosphorylate the right substrate at the right time in human cells remains a key, unanswered question.
The Ly group has applied state of the art mass spectrometry-based proteomics to identify the cell cycle regulated proteome and phosphoproteome (1,2). The group has recently developed a novel phosphoproteomic assay to dissect how non-catalytic subunits of CDKs contribute to substrate choice. 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 showed that non-catalytic subunits of CDK1 can promote phosphorylation of sites lacking the canonical CDK consensus motif, and instead revealed 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 unknown. The PhD project will explore the function of these non-canonical sites and investigate substrate phosphorylation by other CDKs (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 sciences. Additionally, 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.
(1) Kelly et al. Low Cell Number Proteomic Analysis Using In-Cell Protease Digests Reveals a Robust Signature for Cell Cycle State Classification. https://www.mcponline.org/article/S1535-9476(21)00141-9/fulltext
(2) Ly et al. Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS. https://elifesciences.org/articles/27574
Please note that the closing date for this project is Friday 29th April 2022.