Project Outline

Phosphorylation is one of the most highly studied posttranslational modifications, due to its fundamental importance in regulating protein function, interactions, subcellular localisation, and turnover. Protein phosphorylation is also one of the central components of signal transduction pathways within cells through cascades of protein kinase activity. The addition of a phosphate group, PO4, at specific amino acid residues on a protein (typically serine threonine, tyrosine) imparts a localised negative charge that is believed to induce conformational changes or alterations to the surface charge of a protein. Conveniently, phosphorylation also provides an effective means for selective charge-based enrichment of peptides, thus enabling large-scale phospho-proteomics experiments. We now have substantial datasets that have identified many thousands of phosphorylation sites on proteins. However, deriving generally applicable mechanistic knowledge from large phospho-proteomic datasets (PhosPhAt #refs) about how phosphorylation at a specific amino acid governs function has been much slower, due to a need to experimentally validate on an individual basis (#refs Kadota, etc). It would be useful to be able to recognise, prioritise and even predict functionally important phosphorylation sites, in many different systems.

In this project we will focus on the FERONIA pathway because this receptor kinase is involved in many aspects of plant growth, development and immunity, several interacting proteins are known and I have a unique preliminary dataset of FERONIA interactions and downstream phosphorylation events. I hypothesise that FERONIA serves as a hub to integrate stress and growth responses from several different stimuli and unrelated pathways and thus will be a valuable pathway to explore in detail and will provide broadly applicable insights on how protein phosphorylation regulates function through protein interactions. We will leverage 3D protein structure prediction to prioritise specific phosphorylation sites (identified in our preliminary data) for experimental validation in the FERONIA pathway. You will use a combination of bioinformatics, 3D modelling, molecular biology and proteomic methods to identify and validate key functional phosphorylation events.

Techniques

• Genetic manipulation of bacteria to express proteins
• Extraction of proteins from plant and bacteria
• Proteomics and analysis of cross-linking using mass spectrometry
• Protein-protein interaction biochemistry
• Predictive 3D modelling of protein structures
• Mining bioinformatics data resources
• Determining meaning conservation of protein sequences and structures across plant species