Chemical genetic approaches to identifying protein kinase substrates

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Protein phosphorylation is the key regulatory mechanism for many cellular processes. Accurate and timely protein phosphorylation is essential to maintain normal physiology. For example, cell division is regulated primarily by the activities of a family of protein serine/threonine kinases, the cyclin-dependent kinases (cdks); misregulation of these enzymes often leads to aberrant cell division, a situation underlying cancer. Whilst the protein kinases can be simply identified by homology searches, definitive identification of their specific substrates has proved difficult. Current approaches to kinase substrate identification are severely hampered by the low abundance of specific targets amongst the huge background of other phosphoproteins ¿ around 30% of all intracellular proteins are phosphorylated. Almost all protein kinases have a conserved binding site for adenosine triphosphate (ATP) which donates its gamma-phosphate to the substrate protein in the catalysed reaction. Recently, analogues of ATP with bulky moieties introduced at the N6 position have been combined with kinases housing mutationally enlarged ATP binding pockets with the aim of making a unique ATP:kinase pair to facilitate specific radiolabelling of substrates. To date, the major limitation of this approach is that it is only applicable in vitro due to the cell impermeant nature of the ATP analogues. We aim to build on this chemical genetic foundation by developing and applying methodology for the in vivo identification identification the substrates of protein kinases. To achieve this aim we will generate a multifunctional ATP analogue that will self-internalise to enable utilisation by the modified kinases for specific substrate identification. We describe a number of chemical and molecular biological modifications that will enable specific substrate labeling. It is important to stress that the approach we propose can be used to identify the substrates of any protein kinase. As proofs of principle, we will use cdk2 and protein kinase D1 (PKD1) as models upon which to base our methodology but we will also screen a range of other kinases for their suitability. The cdk2 kinase is a key regulator of the G1/S transition and although its centrality has recently been called into question, many cancer cells display deregulated cdk2 activity; PKD is a key intermediate in antigen receptor signal transduction in lymphocytes. Thus, as well as providing our proof of principle, the identification of the substrates of these two kinases is likely to substantially further our understanding of fundamental biological processes and present new targets for therapeutic intervention.