Localized Deactivation Controls Net GTPase Activity

Abstract

Small guanosine triphosphatases (GTPases) are switch proteins that are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). GTPases have an intrinsic GTPase activity that is stimulated by GTPase-activating proteins (GAPs), which limit the activation of their target GTPases. The GTP-bound state of GTPases is promoted by guanosine exchange factors (GEFs) that stimulate the release of GDP in exchange for GTP. Cells typically express multiple GTPases and GEFs and GAPs. Ohba et al. used engineered GTPases that produce a fluorescence resonance energy transfer (FRET) signal when active in combination with conditions that stimulate particular GEFs to show that the differential activation of GTPases at the plasma membrane or internal membranes (predominantly the perinuclear region) could not be explained by compartmentalized delivery or activation of GEFs. That is, localized activation of the GTPases persisted regardless of what GEF was used to stimulate the GTPase, including GEFs activated by soluble signals, such as adenosine 3′,5′-monophosphate (cAMP). When GTPases were mutated so that GAP interactions were blocked, they showed a more diffuse pattern of activation compared with GTPases with normal GAP interaction domains. Spatiotemporal analysis and calculation of the GEF and GAP rate constants suggested that differences in GAP activity in different regions of the cell could be responsible for the localized activation of GTPases. The data from the spatio-temporal analysis was then used to create a cellular simulation for activation of GTPase at the plasma membrane or the internal membranes that reproduced the results seen in the cells. Thus, differential inactivation of the GTPases may allow a uniformly produced activation signal to be restricted to particular subcellular locations. Y. Ohba, K. Kurokawa, M. Matsuda, Mechanism of the spatio-temporal regulation of Ras and Rap1. EMBO J. 22 , 859-869 (2003). [Abstract] [Full Text]