Description
Members of the Ras family of guanine nucleotide binding proteins (GTPases) are nodal regulators of numerous cell biological processes including proliferation, cell-adhesion and apoptosis. Ras cycles between an active GTP-bound state and an inactive GDP-bound conformation. Cellular Ras-GDP/GTP levels are tightly controlled by guanine nucleotide exchange factors (GEFs), which activate Ras by promoting GTP uptake, and GTP hydrolase activating proteins (GAPs), which accelerate conversion of Ras bound GTP to GDP. Traditionally GTPase activity measurements in vivo involved metabolic labelling of cells with inorganic [32P]-phosphate followed by isolation of the GTPase and chromatographic analysis of bound guanine nucleotides. This method provides quantitative data for GDP and GTP levels of Ras but is a tedious and time consuming procedure which requires working with large amounts of radioactivity and is prone to various sources of errors. More recently an alternative non-radioactive technique has been described that exploits the selective interaction of the Ras-binding domain (RBD) of the Ras effector Raf with the active, Ras-GTP conformation. Recombinant, GST-tagged RBD is added to cell extracts to pull out Ras-GTP, which is consequently detected by Western blotting. This approach has greatly accelerated and thus simplified semi-quantitative Ras activity determinations