EMBO Fellows Meeting 2012

John Burke
EMBO Fellows Meeting 2012
Deuterium exchange mass spectrometry used to probe membrane recruitment of
the common oncogene phosphoinositide 3-kinase (p110α)
Abstract
Most cellular responses to extracellular stimuli have a common component of regulation arising from selective
recruitment of a network of signalling complexes to membranes. However, studying these systems remains a
daunting task. We have made unprecedented progress in understanding these systems by applying a
synthesis of deuterium exchange mass spectrometry (DXMS), X-ray crystallography and FRET spectroscopy
towards the PI3 kinase (PI3K) family of proteins. PI3Ks are lipid kinases that are involved in a variety of cellular
functions, including growth, proliferation, and metabolism. The importance of regulating PI3K activity is
highlighted by the fact that the PI3K p110α catalytic subunit (PIK3CA) is one of the most frequently mutated
genes in cancer.
Using DXMS we have examined the activation of wild-type p110α/p85α and a spectrum of oncogenic mutants
in three enzyme states: basal, RTK phosphopeptide activated, and membrane bound. Differences in amide
exchange rates upon activation show that for wild-type p110α/p85α the transition from an inactive cytosolic
conformation to an activated form on membranes entails four distinct conformational events. DXMS results for
cancer mutants show that all upregulate the enzyme by enhancing one or more of these dynamic events.
Protein-lipid FRET and lipid kinase assays showed that all mutations increased binding to membranes and
basal lipid kinase activity, even mutations distant from the membrane surface. Our results elucidate a unifying
mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating motions required for
catalysis on membranes.
John E. Burke, Olga Perisic, Glenn Masson, Oscar Vadas, and Roger L Williams
MRC Laboratory of Molecular Biology, Cambridge UK
14-17 June 2012, Heidelberg, Germany