Most receptor-mediated signal transduction pathways in mammalian cells involve enzymes that generate either soluble or membrane-resident second messengers by modifying phospholipids present in cell membranes. These enzymes are activated in response to a variety of cellular signals such as hormones, cytokines and neurotransmitters.
We are trying to develop a detailed structural understanding of the network of interacting pathways involved in phospholipid signalling. Our current efforts focus on phosphoinositide 3-kinases (PI3Ks) and a group of protein kinases with catalytic domains related to PI3Ks (PIKKs), including mTOR, ATM and ATR. Due to the frequent mutation of these enzymes in human cancers, a structural understanding of the complexes that they make with their regulators is important for drug development.
In addition to X-ray crystallography, electron cryo-microscopy, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and other biophysical methods, we are reconstituting the PI3Ks and PIKKs in vitro with membranes and small G proteins that regulate the complexes. These reconstituted systems give us a clear understanding of the kinetic consequences of structural variants of the enzymes, and they have enabled us to develop high-throughput screens for drug discovery that better mimic the biological processes in which the enzymes participate.