Use of the Ras binding domain of c-Raf for biochemical and live-cell analysis of Ras activation

Small modular GBDs (GTPase-binding domains) derived from GTPase-effector proteins are useful tools for the selective detection of the active GTP-loaded GTPase conformation, be it in biochemical assays or for imaging purposes. Use of GBD probes requires careful consideration of all features of the GDB–GTPase interaction. It is innate to the strong and specific interaction with the GTP-loaded GTPase, that GBDs will protect their partner GTPases from GAP (GTPase-activating protein) action. This feature is likely to cause an increase in cellular Ras-GTP levels, in particular in leucocytes and other cells with high steady-state Ras-GDP/GTP cycling rates. By the same token, high levels of GBD expression will interrupt GTPase-initiated signalling, with implications for the activation of the very same GTPase since feedback regulatory mechanisms can impinge on this process.