BackgroundReceptor tyrosine kinases (RTKs) participate in a multitude of signaling pathways, some of them via the small G-protein Ras. An important component in the activation of Ras is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras.Principal FindingsWe can now demonstrate that the activation of Ras requires, in addition, the essential participation of ezrin, radixin and/or moesin (ERM), a family of actin-binding proteins, and of actin. Disrupting either the interaction of the ERM proteins with co-receptors, down-regulation of ERM proteins by siRNA, expression of dominant-negative mutants of the ERM proteins or disruption of F-actin, abolishes growth factor-induced Ras activation. Ezrin/actin catalyzes the formation of a multiprotein complex consisting of RTK, co-receptor, Grb2, SOS and Ras. We also identify binding sites for both Ras and SOS on ezrin; mutations of these binding sites destroy the interactions and inhibit Ras activation. Finally, we show that the formation of the ezrin-dependent complex is necessary to enhance the catalytic activity of SOS and thereby Ras activation.ConclusionsTaking these findings together, we propose that the ERM proteins are novel scaffolds at the level of SOS activity control, which is relevant for both normal Ras function and dysfunction known to occur in several human cancers.

No abstract

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras.ERM proteins | autoinhibition | GEF regulation T he small GTPase Ras (rat sarcoma) regulates essential cellular processes such as proliferation, motility, and differentiation. Activation of Ras by receptor tyrosine kinases (RTKs) is mediated by the guanine nucleotide-exchange factor (GEF) Son of sevenless (SOS). SOS is recruited by activated RTKs and subsequently engages Ras. In recent years, however, it has been recognized that this simple activation process is subject to a complex regulation. A number of regulatory motifs on SOS have been identified: the C-terminal catalytic Ras-binding domain for nucleotide exchange (1), the N-terminal half that carries histone-like sequences rich in positively charged amino acids, a Dbl homology (DH) domain, and a pleckstrin homology (PH) domain (1, 2). The DH/PH domains decrease the catalytic activity of SOS by folding back on the catalytic domain, thereby restricting accessibility to a second Ras-binding site that is distinct from the catalytic site (2). This allosteric Ras-binding site is important for the activation of SOS. Thus, Ras itself is an essential determinant of SOS regulation (2). Finally, lipid interaction contributes to the activation of SOS: The positively charged histonelike sequences interact with the negatively charged plasma membrane (3, 4). Moreover, binding of both phosphoinositides to the PH domain (5) and phosphatidic acid (PA) to the histone-like domain enhances SOS activity by relieving autoinhibition and exposing the allosteric Ras-binding site (6-8).Our interest in small GTPases was triggered originally by the observation that members of a family of actin-binding proteinsezrin, radixin, and moesin (ERM)-appear to enhance Ras activity (9). We showed that in re...