Abstract
The Ras guanine-nucleotide exchange factor Ras-GRF/Cdc25(Mn) harbors a complex array of structural motifs that include a Dbl-homology (DH) domain, usually found in proteins that interact functionally with the Rho family GTPases, and the role of which is not yet fully understood. Here, we present evidence that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK). In an unprecedented fashion, we have found that these processes are regulated by the Rho family GTPase Cdc42. We show that GDP- but not GTP-bound Cdc42 prevents Ras-GRF recruitment to the membrane and activation of Ras/MAPK, although no direct association of Ras-GRF with Cdc42 was detected. We also demonstrate that catalyzing GDP/GTP exchange on Cdc42 facilitates Ras-GRF-induced MAPK activation. Moreover, we show that the potentiating effect of ionomycin on Ras-GRF-mediated MAPK stimulation is also regulated by Cdc42. These results provide the first evidence for the involvement of a Rho family G protein in the control of the activity of a Ras exchange factor.
Highlights
An inactive, GDP-bound state and an active GTP-bound state, and it is known that this mechanism is controlled by at least two types of regulatory proteins directly acting on Ras: GAPs (GTPase-Activating Proteins), potentiators of the capacity of Ras to hydrolyze GTP, and GEFs (Guanine nucleotide Exchange Factors) that catalyze the exchange of GDP for GTP, promoting its activation [2]
Ras and mitogen-activated protein kinase (MAPK) Activation Induced by Ras-GRF Deletion Mutants—To investigate the role of Ras-GRF DH domain in the activation of the Ras/MAPK pathway, we utilized transient overexpression as an experimental approach, because most of the available data on Ras-GRF biochemistry has been obtained by this method
The different Ras-GRF constructs were cotransfected into COS-7 cells together with an AU5-tagged H-Ras [26], cells were incubated with [P32]orthophosphate, and nucleotide exchange was scored in anti-AU5 immunoprecipitates after thin-layer chromatography
Summary
An inactive, GDP-bound state and an active GTP-bound state, and it is known that this mechanism is controlled by at least two types of regulatory proteins directly acting on Ras: GAPs (GTPase-Activating Proteins), potentiators of the capacity of Ras to hydrolyze GTP, and GEFs (Guanine nucleotide Exchange Factors) that catalyze the exchange of GDP for GTP, promoting its activation [2]. Analysis of Ras-GRF primary structure reveals the presence of a number of functional motifs presumably involved in diverse signaling control mechanisms and protein-protein interactions. Ras-GRF contains a Dbl homology domain (DH) [9] that bears strong resemblance to the oncoprotein Dbl, a GEF for the GTPase Cdc42 [10]. Calcium can regulate Ras-GRF activity by a mechanism mediated through a calmodulin-binding motif (IQ domain) present in its amino terminus [18, 19]. Calcium ionophores like ionomycin can enhance Ras-GRF-mediated activation of the MAPK pathway [19, 20]. Mutations within the DH domain inhibit ionomycin-induced MAPK activation [21], suggesting that the Ras-GRF DH domain may somehow function in the regulation of this signaling route. Regulation of Ras-GRF by Cdc demonstrate that, in an unprecedented fashion, this process is regulated by the GTPase Cdc that when GDP-bound precludes Ras-GRF recruitment to the membrane and the subsequent activation of the Ras/MAPK pathway
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