David Barford - Overview

Molecular basis for the control of cell processes by protein post-translational modification
Nearly all cellular processes are regulated by post-translational modifications such as reversible protein phosphorylation, acetylation and ubiquitin-dependent processes.

These processes regulate the activities of signal transduction cascades that ultimately lead to changes in gene expression and control of the cell cycle and apoptosis. Dis-regulation of these pathways has important implications for the development of cancer. The purpose of our research is to determine the molecular basis for signal transduction cascades by means of X-ray crystallography and electron microscopy.

We are interested in understanding the enzymes that mediate post-translational modifications, and also the structural and functional consequences of these modifications. Understanding these processes at a molecular level, as defined by their structural, kinetic and thermodynamic properties will be key to the development of novel cancer therapies.

Reversible protein phosphorylation underlies the regulation of nearly all cellular processes. Nearly one-third of all intracellular proteins are phosphorylated and the protein kinases and phosphatases that regulate the overall levels of protein phosphorylation constitute a significant proportion of the human genome. We have characterised the structures of a number of protein phosphatases and are now studying those that have direct relevance to cancer, for example PP5 and Cdc14 that negatively regulate the tumour suppressor gene product p53, and which also regulate cell cycle progression. In addition, we are interested in the mechanism of the specific dephosphorylation of Thr and Tyr residues of the MAP kinase ERK2 by MKP3, and efforts are underway to crystallise the kinase-phosphatase complex.

Many protein kinases positively regulate cell growth and proliferation and are proto-oncogenes. One such protein, protein kinase B (PKB/Akt), is a proto-oncogene that mediates cell survival and growth and inhibits cell apoptosis, and is stimulated in response to activation of PI-3 kinase and generation of phosphatidyl inositol 3,4,5 tris-phosphate. Elevated PKB activity is implicated in numerous cancers including breast, prostate, melanomas and glioblastomas. By determining the crystal structure of PKB we aim to develop specific inhibitors of the enzyme that will be useful in the treatment of cancer. Similarly, we aim to understand the structures of the Raf-kinase and PDK1.

Many cellular processes, particularly the cell cycle and signal transduction pathways, are controlled by selective protein degradation in a mechanism that is mediated by ubiquitin-dependent proteolysis by the proteosome. The selectivity of this process is determined by a class of proteins termed E3 protein ubiquitin ligases that catalyse the poly-ubiquitination of target proteins. We are studying the anaphase promoting complex (APC) that regulates cell cycle progression at the metaphase to anaphase transition, and at the exit from mitosis. The APC is a multi-subunit complex of 11 core subunits and a variable 12th co-activator subunit that functions to determine substrate selectivity. We aim to determine the conformation of the APC from various stages of the cell cycle by electron microscopy and X-ray diffraction, and correlate these structures to defined biological activities. Finally, we would like to understand the molecular mechanism of how protein phosphorylation and acetylation regulates p53 activity.