SOS (Son of Sevenless) is the principal Ras guanine nucleotide exchange factor downstream of receptor tyrosine kinases, and its activity is interdependent with its product: SOS loads GTP onto Ras, while Ras•GTP in turn binds a distal allosteric site on SOS and stimulates its catalytic activity, creating a positive feedback loop gated by receptor-driven membrane recruitment and release of SOS autoinhibition. The molecular pieces of this loop are established piecewise — structurally, biochemically, and in reconstituted membrane systems — and the cellular phenomena they produce (feedback-driven digital and hysteretic Ras activation, endocytic removal of SOS after each signaling round, gating by phase-separated scaffold clusters) are experimentally described. The primary source of this problem, the 2019 review by Bandaru, Kondo, and Kuriyan, synthesizes these results and identifies the structural and mechanistic questions that remain unresolved. The problem is to resolve those open questions: the basis of interconversion between the highly and lowly active membrane-anchored SOS states, how the SOS C-terminal (proline-rich) tail weakens Ras•GTP affinity at the allosteric site, whether SOS prefers Ras•GDP over Ras•GTP as substrate, and whether REM domains of other Ras-specific GEFs interact with as-yet-unidentified allosteric regulators.

Background

Ras proteins are small regulatory GTPases that cycle between an inactive GDP-bound state and an active GTP-bound state and sit at the top of the MAPK (mitogen-activated protein kinase) signaling cascade that controls proliferation and differentiation in metazoan cells. Ras is switched on by guanine nucleotide exchange factors (GEFs), which pry open the nucleotide pocket so that fresh GTP can load, and switched off by GTPase-activating proteins (GAPs), which accelerate GTP hydrolysis.

Son of Sevenless (SOS) is the main Ras-specific GEF acting downstream of receptor tyrosine kinases (RTKs). SOS is a large multi-domain enzyme: N-terminal histone-like and DH (Dbl homology)–PH (pleckstrin homology) domains that hold the catalytic core in an autoinhibited conformation, followed by a REM (Ras exchange motif) domain and a cdc25 domain that together form the catalytic unit. Besides the catalytic site, the REM–cdc25 unit carries a second, distal site that binds Ras allosterically. Structural and biochemical work (Margarit et al. 2003) showed that binding of Ras•GTP — the very product of SOS activity — at this distal allosteric site strongly stimulates SOS catalytic activity: Ras•GTP stabilizes the active geometry of the catalytic site, roughly an order of magnitude in exchange rate. Because SOS is recruited to activated RTKs at the plasma membrane through the adaptor protein Grb2, and Ras•GTP is membrane-anchored, the product of SOS feeds back onto SOS itself: a positive feedback loop in which Ras•GTP activates its own activator.

Cellular and single-molecule work mapped the loop piecewise. Feedback shapes Ras signaling dynamics in cells (Boykevisch et al. 2006); Ras activation in lymphoid cells is digital and hysteretic, arising from interplay between two GEF systems, RasGRP1 and SOS, with SOS feedback supplying the all-or-none character (Das et al. 2009). On supported lipid membranes, allosteric Ras•GTP activates SOS by altering the enzyme's dynamic fluctuations, and one membrane-bound SOS molecule processively turns over hundreds of Ras molecules before detaching (Iversen et al. 2014; Gureasko et al. 2008). SOS is removed from the membrane by endocytosis after each signaling round (Christensen et al. 2016), so sustained Ras output requires continuous recruitment.

The primary source of this problem, a 2019 review by Bandaru, Kondo, and Kuriyan ('The Interdependent Activation of Son-of-Sevenless and Ras'), synthesizes these results. The molecular cycle — membrane recruitment, conformational opening when autoinhibition is released, and occupation of the allosteric site by Ras•GTP — has been mapped mostly in purified or reconstituted systems, and the review treats the loop's cellular manifestations (digital/hysteretic Ras activation, endocytic removal of SOS, gating by scaffold clusters) as established findings. The review's explicitly open items are: the structural basis for interconversion between the highly and lowly active states of membrane-anchored SOS is 'not yet understood'; the structural details of how the SOS C-terminal (proline-rich) tail weakens Ras•GTP affinity at the allosteric site are 'unknown' (Lee et al. 2017); whether SOS prefers Ras•GDP over Ras•GTP as substrate 'needs further investigation', because the existing conclusion was not based on a direct comparison of the rates of nucleotide exchange for Ras•GDP and Ras•GTP by SOS; and whether the REM domains of other Ras-specific GEFs modulate activity by interacting with as-yet-unidentified allosteric regulators is 'an intriguing question for further investigation'. A second source review (Quilliam 2007) frames the same architecture — multiple regulatory domains of SOS controlling the duration and amplitude of Ras activation — from the earlier literature.

Problem Statement

Resolve the open structural and mechanistic questions that the primary source (Bandaru, Kondo & Kuriyan 2019) identifies in the interdependent SOS–Ras activation loop: (a) the structural basis of interconversion between the highly and lowly active states of membrane-anchored SOS, which the review states is 'not yet understood'; (b) the structural details of the mechanism by which the SOS C-terminal (proline-rich) tail weakens the affinity of Ras•GTP for the allosteric site of SOS, which the review states are 'unknown'; (c) whether SOS prefers Ras•GDP over Ras•GTP as substrate, which 'needs further investigation' because the existing conclusion was not based on a direct comparison of the rates of nucleotide exchange for Ras•GDP and Ras•GTP by SOS; and (d) whether the REM domains of other Ras-specific GEFs modulate activity by interacting with as-of-yet unidentified allosteric regulators. The four parts (a)-(d) are independently posed: determining the single preference in (c) does not by itself address (a), (b), or (d). These questions concern the loop described in the Background — receptor-driven recruitment of SOS to the plasma membrane (via Grb2 and scaffold-protein clustering), release of SOS autoinhibition and the associated conformational opening of the catalytic core, and occupation of the distal allosteric site by Ras•GTP — whose interdependent activation sets the amplitude and duration of the Ras response and whose mechanistic mapping has been achieved mostly in purified or reconstituted systems.

References

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