Protein phosphorylation dynamics and cell division control in human cells

Interested individuals must follow Steps 1, 2 and 3 at this link on how to apply
http://www.ed.ac.uk/biology/prospective-students/postgraduate/pgr/how-to-apply

The temporal regulation of protein abundance and post-translational modifications is a key feature of mitosis. Mitosis is characterized by ordered progression through several subphases, e.g. prophase, prometaphase, metaphase, anaphase, and telophase, that are distinguished by substantial differences in cell structure. These major cellular state changes are driven at the molecular level by regulatory mechanisms that include protein phosphorylation and dephosphorylation. A major challenge with the biochemical analysis of protein phosphorylation during mitosis in human cells is that each mitotic subphase is exceedingly short, typically lasting only minutes, with cell-to-cell variability in dwell times.

Chemical synchronization, e.g. via spindle poisons or small molecule kinase inhibitors, can be used to enrich for mitotic populations but with major caveats. Chemical genetic approaches aim to reduce artefacts associated with non-specific effects of small molecule inhibitors by genetically engineering endogenous kinases to have a larger ATP binding pocket that accommodates PP1-analogues, known as analogue sensitive (AS) mutants. Addition of PP1-analogues selectively inhibits AS mutants. Previously, yeast and DT40 cells have been synchronized at the G2/M border by replacing endogenous cyclin dependent kinase 1 (CDK1) with a CDK1 AS mutant and treatment with PP1-analogues. This project will exploit a human (HeLa) cell line with an analogue-sensitive CDK1, which enables us to obtain highly enriched populations of cells entering mitosis in a synchronous wave.

Recent advances in mass spectrometry-based proteomics, the combination of fluorescence-activated cell sorting with proteomics, and chemical genetics, affords new opportunities to revisit the biochemistry of mitosis with unprecedented temporal resolution.

PhD research aims

The interdisciplinary PhD project aims to dissect phosphorylation dynamics during mitosis in human cells in three parts:

1. High temporal resolution map of protein phosphorylation during cell division in human cells
• Combine chemical genetic approaches and fluorescence-activated cell sorting to isolate ‘pure’ subpopulations of mitotic subphases defined by biochemical state
• State of the art mass spectrometry-based phosphoproteomics to measure differences in protein phosphorylation

2. Understanding dynamic control by phosphatases & kinases
• Engineer human cell lines that enable inducible inhibition of protein kinases and/or phosphatases using CRISPR/Cas9-mediated recombination
• Measure kinetics of phosphorylation and dephosphorylation by individual kinases and phosphatase classes by using chemical inhibitors and phosphoproteomics

3. Bioinformatic analysis
• Identify sequence motifs from analysis of phosphorylation sequences and dynamics
• Modelling contribution of individual kinase and phosphatase activities to endogenous phosphorylation dynamics

Training

The PhD research project will provide expert training opportunities in human cell culture, cell line engineering, flow cytometry, protein biochemistry, quantitative proteomics and phosphoproteomics, and the handling and analysis of large scale phosphoproteomic data.


Further Information

The successful PhD student will be jointly supervised by Dr Tony Ly, a Sir Henry Dale Fellow, and Prof Bill Earnshaw, FRS, FRSE, FMedSci. Dr Ly and Prof Earnshaw are group leaders in the Wellcome Centre for Cell Biology (WCCB). The WCCB is one of eight UK-based Wellcome Trust Centres, two of which are in Scotland. The PhD research will be supported by funding from the Wellcome Trust and the Royal Society.