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.
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Institute of Molecular Cell Biology, Medical Faculty, Friedrich-Schiller-University Jena, Drackendorfer Str.1, 07747 Jena, Germany. b5igru@rz.uni-jena.de
D000818:Animals; D001665:Binding Sites; D006801:Humans; D007962:Leukocytes / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D020298:U937 Cells; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D020703:ras GTPase-Activating Proteins; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins