Search

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation., Competing Interests: Declaration of interests All authors declare they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Membrane-anchored Ras family proteins are activated by guanine nucleotide exchange factors such as SOS1. The CDC25 domain of SOS1 catalyzes GDP-to-GTP exchange, thereby activating Ras. Here, we aim to decipher the activation mechanism of KRas4B, a significantly mutated oncogene. We perform large-scale molecular dynamics simulations on 12 SOS1 systems, scrutinizing each step in two possible KRas4B activation cycles, fast and slow. To activate KRas4B at the CDC25 catalytic site, the allosteric site in the Ras exchanger motif (REM) domain of SOS1 needs to recruit a (nucleotide-bound) KRas4B molecule. Our simulations indicate that KRas4B-GTP interacts with the REM allosteric site more strongly than with the CDC25 catalytic site, consistent with its allosteric role in the GDP-to-GTP exchange. In the fast cycle, the allosteric KRas4B-GTP induces conformational change at the catalytic site. The conformational change facilitates loading KRas4B-GDP at the catalytic site and opening the KRas4B nucleotide-binding site for GDP release and GTP binding. GTP binding reduces the affinity of KRas4B-GTP to the CDC25 catalytic site, resulting in its release. By contrast, in the slow cycle, KRas4B-GDP binds at the allosteric REM site. The limited, altered conformational change that it induces prevents the exact alignments of switch I and II of KRas4B. The increasing binding strength at both binding sites due to interactions of regions other than switch I and II retards GDP release from the catalytic KRas4B, thus KRas4B activation. The accelerated activation cycle supports a positive feedback loop with allosteric signals communicating between the two Ras molecules and is the predominant, native function of SOS. SOS1 activation details may help drug discovery to inhibit Ras activation., (Copyright © 2018 Biophysical Society. All rights reserved.)

Ligand-induced receptor aggregation is a well-known mechanism for initiating intracellular signals but oligomerization of distal signaling molecules may also be required for signal propagation. Formation of complexes containing oligomers of the transmembrane adaptor protein, linker for the activation of T cells (LAT), has been identified as critical in mast cell and T cell activation mediated by immune response receptors. Cross-linking of LAT arises from the formation of a 2:1 complex between the adaptor Grb2 and the nucleotide exchange factor SOS1, which bridges two LAT molecules through the interaction of the Grb2 SH2 domain with a phosphotyrosine on LAT. We model this oligomerization and find that the valence of LAT for Grb2, which ranges from zero to three, is critical in determining the nature and extent of aggregation. A dramatic rise in oligomerization can occur when the valence switches from two to three. For valence three, an equilibrium theory predicts the possibility of forming a gel-like phase. This prediction is confirmed by stochastic simulations, which make additional predictions about the size of the gel and the kinetics of LAT oligomerization. We discuss the model predictions in light of recent experiments on RBL-2H3 and Jurkat E6.1 cells and suggest that the gel phase has been observed in activated mast cells.