Functional proteomics analysis of GTPase signaling networks

Abstract

This chapter discusses the functional proteomics analysis of GTPase signalling networks. Posttranslational modifications of GTPase have been found to be an essential component in GTPase action. Protein kinases are critical upstream and downstream regulators of GTPase signals. Thus, sensitive methods to assess targets of lipid modification, kinase levels, activation, and phosphorylation sites are required. Most importantly, multiprotein complexes, not individual proteins, are the most physiologically relevant mediators of signal transduction. Therefore, methods are needed to isolate native complexes from biologically appropriate cells and to identify the individual components. The chapter also describes the technique of farnesyltransferase inhibitors (FTIs) and prometic analysis. Potential applications of proteomics technology in Ras family biology include (1) determining the proteins that are activated in a given cell type after Ras transformation, including both immediate downstream effectors and elements further downstream not detectable by yeast two-hybrid, coimmunoprecipitation, or other analyses), (2) ascertaining the particular set of GEF (GTP/GDP exchange factor)/GTPase pairs or GTPase/effector pairs interacts in a given cell, (3) identifying individual components in a multiprotein signaling complex, including both signaling and scaffolding proteins, and (4) determining the consequences of inhibiting the function of Ras family or other signaling molecules to identify their mechanism of action. The chapter concludes with a a discussion on analysis of proteins from two-dimensional polyacrylamide.