In-depth quantification and characterisation of PI 3kinase signalling networks

We work on a group of signal transduction proteins called phosphoinositide 3-kinases (or PI3Ks in short) that play important roles in many biological functions in the healthy organism, and are also implicated in diseases such as diabetes, allergy, and cancer. There are 8 different PI3K genes and it is becoming clear that they have different functions and that they are involved in different diseases. The delineation of the roles of PI3K genes not only has purely academic importance in providing insight into fundamental biological processes but is also of huge interest for the ongoing development of drugs that inhibit specific forms of PI3Ks to treat different diseases. Despite many recent scientific advances in the field, there is little information on how the different forms of PI3K perform their distinct biological functions. The hypothesis we would like to test is that the different PI3Ks differ in the way by which they affect a group of proteins called protein kinases and their substrates. To test this hypothesis we will use a novel method that we have developed in the laboratory, based on a technique known as mass spectrometry, to quantify how active protein kinase pathways are in cells. The advantage of mass spectrometry is that it analyses the activities of protein kinases in a comprehensive fashion, allowing to quantify thousands of activities simultaneously. This depth of analysis is allowing us to quantify protein kinase activity without preconceptions of which of the many kinases in cells may or may not be affected by PI3K. It therefore also allows to discover unknown mechanisms of PI3K signal transduction. In the work proposed in this application we will inactivate the different PI3Ks in cells by pharmacological and genetic means, and compare to normal or untreated cells. The focus of this work will be on 3 different PI3Ks known as p110alpha, p110beta and p110delta. These results are likely to lead to the discovery of pathways specifically affected by the different PI3K genes. These results would be of high impact in the field and also allow to discover markers for drugs that inhibit these PI3Ks selectively. The second stage of the project involves investigating the role of these newly identified proteins as potential players in the function of PI3K, which will be achieved by standard genetic and biochemical approaches in cell-based studies.

This proposal offers a timely opportunity of combining unique expertise in analytical cell signalling and PI3K biology, now that the required technologies and genetic and pharmacological tools have matured to a level that can be used to start to address key questions in the field. There are 8 catalytic isoforms of PI3K and the creation of gene-targeted mice and pharmacological inhibitors with specificity for the different isoforms are contributing to the understanding of the biological functions of the different isoforms. Despite this information, little is known about the mechanisms by which distinct PI3K isoforms exert their non-redundant functions. The aim of this proposal is to investigate how inactivation of specific isoforms of the class IA subset of PI3Ks affects the activity of downstream protein kinases. For this, we will use a technique, based on mass spectrometry, for in-depth quantification of kinase activities. This method quantifies peptides bearing sites of phosphorylation. Our preliminary data indicate that it is possible to quantify thousands of phosphorylated sites with time frames compatible with the use of the technique as a 'routine' readout of biological experiments. Cells will be exposed to pan and isoform-selective PI3K inhibitors; phosphorylated sites will then be quantified with our techniques for in-depth analysis of kinase activities. Other experiments will involve comparing kinase activities in fibroblasts with genetic inactivation of Class IA PI3K isoforms with their wild-type counterparts. The second stage of the project involves validation of the mass spectrometry experiments using standard biochemical techniques and to address the role of selected novel PI3K pathway members in signalling and cell biology. These experiments are likely to provide novel information on how the PI3K pathway operates and could lead to the identification isoform-specific PI3K signalling pathways for the first time.