Development of new tools for kinomics

This short term project aims to explore the viability of azidophosphate-based systems as tools for unravelling kinase-based signalling systems. Signals in biological systems are communicated via a process known as phosphorylation. In this process enzymes known as kinases pass on signals by transferring a small chemical entity (the phosphate group) from a molecule known as ATP to a signalling protein (kinase substrate). Once a protein has been phosphorylated, and only when it has been phosphorylated, it can bind to another receptor protein (phospho-protein recptor) and thus pass on the signal. Chemical and genetic methods have been developed for the identification kinase substrates, however the identification of phosphoprotein receptors is more challenging. Genetic methods have been devised, but these are complicated. Chemical methods are currently not available and we aim to fill this technology gap. All biological systems contained phosphorylation-mediated signalling pathways thus this new technology has a very broad potential user base which ranges from biomedical researches to basic science researchers.

The proposed work represents an early stage (pilot) investigation of a new tool for understanding phosphorylation-based signalling pathways. We will explore the possibility of using azide-labelled phosphate as a tool for identifying receptors that bind to phosphorylated proteins and peptides. We will determine whether the azide-labelled phosphoryl group of gamma-azido-ATP is transferred to known substrate peptides in a well-characterised system: we will initially use protein kinase A (PKA) with the known peptide substrate (LRRASLG). This will be accomplished using LC and MS techniques. Gamma-azido-ATP will be synthesised and purified using a literature strategy. We will explore the photocross-linking properties of azidophosphorylated proteins for use in the identification of downstream signalling elements i.e. identify proteins that bind only to phosphorylated-substrates. Azidophosphorylated peptide substrate (e.g. LRRASLG) will be photolysed in the presence of anti-phosphosubstrate-specific antibodies that are commercially available. The reactive nitrene species that will be generated via photolysis will react with any proximal protein residues either within the substrate itself or, more importantly with proximal antibody residues. Electrophoresis will be used to determine whether covalent bonding between azidophosphorylated-LRRASLG and the antibody has occurred. Flurophore-labelled-LRRASLG will be used for deteection purposes. We will explore the bioconjugation of azidophosphorylated substrates using click chemistry. Bioconjugation using biotinylated alkynes should allow pull-down purification of the azidophosphorylated species thus facilitating subsequent identification. We will use azidophosphorylated substrate for this purpose and MS techniques to prove that conjugation has occurred. The pH stability of the triazole-phosphate group will also be investigated so that suitable capture and release strategies can be devised, again LC and MS will be used.