Chemical genetic approaches to identifying protein kinase substrates
Lead Research Organisation:
Imperial College London
Department Name: Biological Sciences
Abstract
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Technical Summary
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.
Publications
Bonnac L
(2010)
Synthesis and O-phosphorylation of 3,3,4,4-tetrafluoroaryl-C-nucleoside analogues.
in Organic & biomolecular chemistry
Child ES
(2011)
Inhibition of mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 2 (Cdk2) by platinum(II) phenanthroline complexes.
in Journal of chemical biology
Child ES
(2010)
A cancer-derived mutation in the PSTAIRE helix of cyclin-dependent kinase 2 alters the stability of cyclin binding.
in Biochimica et biophysica acta
Elphick LM
(2009)
Exploring the roles of protein kinases using chemical genetics.
in Future medicinal chemistry
Elphick LM
(2007)
Using chemical genetics and ATP analogues to dissect protein kinase function.
in ACS chemical biology
Elphick LM
(2009)
A quantitative comparison of wild-type and gatekeeper mutant cdk2 for chemical genetic studies with ATP analogues.
in Chembiochem : a European journal of chemical biology
Lee SE
(2011)
The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.
in Chembiochem : a European journal of chemical biology
Lee SE
(2009)
Synthesis and reactivity of novel gamma-phosphate modified ATP analogues.
in Bioorganic & medicinal chemistry letters
Wang N
(2015)
Clickable 5'-?-Ferrocenyl Adenosine Triphosphate Bioconjugates in Kinase-Catalyzed Phosphorylations
in Chemistry - A European Journal
Description | we developed novel methods for identifying the substrates of a class of enzymes called protein kinases. These enzymes are central to many human diseases. Substrate identification was partially achieved using a combination of chemistry and molecular biology |
Exploitation Route | further work could develop the methodology for identification of protein kinase substrates |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | The data provided several collaborations which are on going and a variety of publications |
First Year Of Impact | 2007 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | Project Grant |
Amount | £30,000 (GBP) |
Organisation | Cancer and Polio Research Fund |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2015 |
End | 08/2017 |
Description | 2 |
Organisation | Japanese Foundation for Cancer Research |
Country | Japan |
Sector | Hospitals |
PI Contribution | Joint publication |
Collaborator Contribution | Joint Publicaiton |
Impact | Joint publication |
Start Year | 2006 |
Description | princes trust |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | talks to newly qualified teachers talking a course run by the Prince's Trust |
Year(s) Of Engagement Activity | 2009,2010,2011,2012,2013,2014,2015 |
Description | school visit |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Enthusing school children with science - biochemistry |
Year(s) Of Engagement Activity | 2014 |