Mechanisms through which Sos-1 coordinates the activation of Ras and Rac

Abstract

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1–Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1–Grb2 (S/G) or a Sos-1–E3b1–Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1–Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

“…This is similar to the mechanism of stimulation of Sos exchange activity. The Sos DH domain is active for Rac1 exchange only when Sos is in a ternary complex with Eps8 and E3b1 (Scita et al, 1999(Scita et al, , 2001Innocenti et al, 2002). We investigated whether binding of interacting proteins to the SH3 domains can derepress the exchange activity in vitro.…”

“…As in the case for the Vav GEF, intersectin 1L may need to be phosphorylated in order to derepress the DH domain (Crespo et al, 1997;Han et al, 1997;Schuebel et al, 1998;Aghazadeh et al, 2000). Similarly, binding of an additional protein in vivo may be necessary, as in the case of activation of Sos Rac1 exchange activity by formation of a ternary complex (Scita et al, 1999(Scita et al, , 2001Innocenti et al, 2002). Because our studies have utilized recombinant proteins that are missing the amino-terminal domains, a third possibility is that a missing amino-terminal component of intersectin 1L is involved in the regulation of exchange activity.…”

“…The noncatalytic parts of the structurally complex GEFs link the exchange activity to cellular processes and inhibit the DH domain exchange activity (Zheng, 2001;Hoffman and Cerione, 2002). Cellular inputs, such as protein (Hart et al, 1998;Scita et al, 1999;Innocenti et al, 2002) and phospholipid binding to (Han et al, 1998;Nimnual et al, 1998;Crompton et al, 2000;Das et al, 2000;Russo et al, 2001), and phosphorylation of (Crespo et al, 1997;Han et al, 1997;Schuebel et al, 1998;Aghazadeh et al, 2000) these regulatory regions derepress exchange activity. Integration of cellular signals by Rho GEFs can focus GTPase activity to allow temporal and spatially localized activation of actin cytoskeletal rearrangements.…”

N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits

“…Constructs for EGFP-tagged N-WASP, WASP, WAVE2, WIP, Nck1/2 and Grb2 were as described previously (Benesch et al, 2002;Lommel et al, 2001;Scaplehorn et al, 2002). Expression constructs for myc-tagged Scar-WA and Scar-W (Machesky and Insall, 1998) were kindly provided by Laura Machesky (Birmingham, UK); mRFP-WAVE2 was from Julia Ehinger (Braunschweig, Germany) and EGFP-Sos-1 (Innocenti et al, 2002) was kindly provided by Giorgio Scita (Milan, Italy).…”

The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

“…Sos is a Ras/Rac guanine nucleotide exchange factor (GEF) that associates with the Slit-bound Robo receptor in a ternary complex with the SH3-SH2 adaptor protein Dreadlocks to regulate Rac activity (Yang and Bashaw, 2006). Studies in mammalian cells show that the Rac-GEF activity of Sos is highly dependent upon its precise subcellular localization (Innocenti et al, 2002), suggesting that if Sos recruitment to the plasma membrane was blocked, its activity would be disrupted. In order to determine whether Kuz regulates the Slit-dependent recruitment of Sos to the plasma membrane, we used the mammalian HEK293T system.…”