The Ras activator SOS carries an allosteric pocket that binds RasGTP, creating a positive feedback loop that Das et al. (Cell, 2009) proposed underlies digital (all-or-none), hysteretic Ras–ERK activation with short-term signaling memory in lymphocytes, with the second Ras guanine nucleotide exchange factor RasGRP1 supplying analog (graded) activation. The authors explicitly flagged as unresolved: direct genetic demonstration in primary mammalian lymphocytes in vivo that the SOS allosteric-pocket feedback is the operative mechanism; how compartmentalized Ras signaling and Ras nanoclustering interact with SOS feedback; how downstream MAPK feedback loops and phosphatases shape measured digital/hysteretic readouts; the molecular determinants and timescales of the short-term memory; and the in vivo significance for T-cell antigen discrimination, serial-encounter signal integration, and thymic selection. Seventeen years of follow-up work has reconstituted the SOS–Ras module in vitro — revealing that bimodal Ras activation can arise from SOS processivity without deterministic bistability, with no hysteresis in reconstitution — and produced conflicting evidence in primary human T cells, but the in-vivo genetic test and functional demonstration remain undone. The open problem is to establish or refute the SOS-feedback mechanism of digital Ras signaling in primary lymphocytes in vivo and determine its functional consequences for lymphocyte decisions.

Background

Ras proteins are small GTP-binding molecular switches that cycle between an inactive GDP-bound and an active GTP-bound state; in T and B lymphocytes, engagement of the antigen receptor (the T-cell receptor, TCR, or B-cell receptor) activates Ras, which then switches on the RAF–MEK–ERK protein kinase cascade (a MAPK pathway) to drive functional decisions. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), which accelerate GDP release so that GTP can rebind. Lymphocytes express two families of RasGEFs with distinct regulation: SOS, which is recruited to the plasma membrane through the adaptor protein Grb2 binding to tyrosine-phosphorylated scaffold proteins such as LAT, and RasGRP1, which is recruited and activated by diacylglycerol and calcium — second messengers generated by phospholipase Cγ1 (PLCγ1) after receptor stimulation — and can act on internal membranes as well as the plasma membrane.

SOS is unusual among RasGEFs in possessing an allosteric pocket, formed between its REM and cdc25 domains and distinct from the catalytic site: binding of RasGTP to this pocket stabilizes SOS in an active conformation and markedly increases its catalytic activity. Because SOS produces the very molecule that activates it, SOS constitutes a positive feedback loop; structural and single-molecule work has additionally shown that, once active at the membrane, a single SOS molecule can processively activate thousands of Ras molecules before detaching (Bandaru et al., 2019). Strong positive feedback can in principle make a signaling system bistable — endowed with two stable steady states separated by a threshold — which manifests as digital (all-or-none, bimodal) single-cell responses, hysteresis (the dose–response for activation differs depending on whether stimulus is being increased or decreased, so activation can persist at stimulus levels that could not initiate it), and short-term molecular memory of past stimulation. In the same cells, RasGRP1 acting alone produces analog (graded) responses whose amplitude tracks stimulus strength.

The functional stakes of this distinction are high: T cells must discriminate between antigenic and self peptide–MHC ligands (the complexes of peptide and major histocompatibility complex proteins recognized by the TCR) that can differ by a single amino acid, and Altan-Bonnet and Germain (2005) had proposed that feedback control of digital ERK responses contributes to this discrimination. Das et al. (2009) combined stochastic simulations of the membrane-proximal Ras network with experiments in Jurkat T-cell and DT40 B-cell lines (including SOS1/SOS2-deficient cells reconstituted with wild-type or mutant SOS) and primary lymph-node T cells. They reported that single-cell phospho-ERK distributions are bimodal (digital) when SOS is present but graded when RasGRP1 acts alone; that population-level RasGTP dose–response curves show SOS-dependent hysteresis; and that Ras activation retains short-term memory during serial stimulation, so a cell recently stimulated responds to a weaker second stimulus. Importantly, single-cell assay of RasGTP itself was technically impossible at the time, so bistability was inferred from population-level hysteresis rather than directly observed, and the authors noted that downstream MAPK feedback could confound readouts such as ERK phosphorylation and the CD69 activation marker. A companion computational study (Prasad et al., 2009) showed that such a digital Ras response could generate the sharply defined ligand-potency boundary of negative selection in the thymus (deletion of self-reactive thymocytes), while positive selection (survival and maturation) proceeds via graded responses.

A further spatial dimension comes from the organization of active Ras into nanometer-scale plasma-membrane domains (nanoclusters), which are required for high-fidelity Ras-to-ERK transmission (Tian et al., 2007). In their Discussion, Das et al. explicitly identified five unresolved issues: the lack of a direct genetic demonstration of the SOS feedback mechanism in primary lymphocytes (requiring appropriate mouse models or targeted SOS allosteric-pocket mutants); how analog and digital Ras activation are organized in time and space, and whether Ras nanoclustering is influenced by the positive feedback; how MAPK-pathway feedback loops and phosphatases modulate or confound the measured digital and hysteretic outputs; the determinants and timescales of the feedback-conferred short-term memory; and the in-vivo significance of SOS-dependent digital signaling and hysteresis for antigen discrimination, integration of serial encounters with antigen-presenting cells, and thymic-selection thresholds. These linked issues, updated by what later work has and has not settled, constitute the present problem.

Problem Statement

Das et al. (2009) established, in cell lines and population-level assays, that the positive feedback loop formed when RasGTP binds the allosteric pocket of the Ras guanine nucleotide exchange factor (RasGEF) SOS produces digital (all-or-none), hysteretic Ras–ERK activation with short-term molecular memory in lymphocytes, while RasGRP1-mediated activation is analog — and they explicitly identified as unresolved the direct proof of this mechanism in primary lymphocytes in vivo and its functional consequences. The problem, with one common objective — to determine whether the SOS allosteric-feedback module is the operative mechanism generating digital Ras signaling in primary lymphocytes, and what functional consequences this has for lymphocyte decisions — comprises the following linked questions, none of which later literature has closed:

(1) Mechanism in primary lymphocytes in vivo: directly demonstrate — or refute — that SOS positive feedback via its allosteric pocket is the operative mechanism producing digital Ras–ERK activation in primary mammalian lymphocytes. This test is sharpened by conflicting evidence that TCR-induced ERK activation in primary human T cells does not require Grb2/SOS at all (Warnecke et al. 2012), and by reconstitution data showing bimodal Ras activation without deterministic bistability (Lee et al. 2024).

(2) Spatial determinants: determine how compartmentalized Ras signaling (SOS acting at the plasma membrane versus RasGRP1 also on internal membranes) and the nanoclustering of active Ras interact with SOS feedback and RasGRP1 to shape the temporal and spatial analog/digital response modes in intact lymphocytes.

(3) Feedback integration and readout confounds: quantify how MAPK-pathway feedback loops and phosphatases modulate or confound SOS-dependent digital outputs and hysteresis in measured readouts such as ERK phosphorylation and CD69 — including resolving the inhibitor dose- and timing-dependent puzzles noted in the source paper — and distinguish SOS-level bistability/hysteresis from downstream analog-to-digital conversion.

(4) Memory determinants and timescales: identify the molecular determinants and timescales governing the duration of the short-term memory conferred by SOS feedback — including how long RasGTP occupies the SOS allosteric site, and how Grb2/SOS recruitment dynamics, SOS membrane retention, and SOS removal (e.g., by endocytosis) control the decay of Ras activation between serial encounters.

(5) Functional significance in vivo: test whether SOS-dependent digital signaling and hysteresis matter for T-cell antigen discrimination, integration of serial encounters with antigen-presenting cells, and the sharp positive/negative selection boundary in the thymus.

The problem is answered when the SOS-feedback mechanism of digital Ras activation is demonstrated or refuted in primary lymphocytes in vivo (component 1), and its functional consequences for lymphocyte activation, signal integration, and thymic selection are established or excluded (component 5). Components 2–4 — spatial organization, feedback integration and readout confounds, and memory determinants — remain linked source-flagged open directions that inform the interpretation of components 1 and 5, rather than separate deliverables required for completion. A genuine refutation — for instance, normal digital Ras signaling and selection in feedback-deficient animals, or a demonstrated alternative mechanism for digital Ras signaling in primary lymphocytes — would equally resolve the problem.

References

  1. Das J, Ho M, Zikherman J, Govern C, Yang M, Weiss A, Chakraborty AK, Roose JP. Digital signaling and hysteresis characterize Ras activation in lymphoid cells. Cell 136(2):337-351, 2009. DOI: 10.1016/j.cell.2008.11.051. https://doi.org/10.1016/j.cell.2008.11.051
  2. Jun JE, Yang M, Chen H, Chakraborty AK, Roose JP. Activation of extracellular signal-regulated kinase but not of p38 mitogen-activated protein kinase pathways in lymphocytes requires allosteric activation of SOS. Molecular and Cellular Biology 33(12):2470-2484, 2013. DOI: 10.1128/MCB.01593-12. https://doi.org/10.1128/MCB.01593-12
  3. Warnecke N, Poltorak M, Kowtharapu BS, Arndt B, Stone JC, Schraven B, Simeoni L. TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells. EMBO Reports 13(4):386-391, 2012. DOI: 10.1038/embor.2012.17. https://doi.org/10.1038/embor.2012.17
  4. Kortum RL, Sommers CL, Alexander CP, Pinski JM, Li W, Grinberg A, Lee J, Love PE, Samelson LE. Targeted Sos1 deletion reveals its critical role in early T-cell development. Proceedings of the National Academy of Sciences USA 108(30):12407-12412, 2011. DOI: 10.1073/pnas.1104295108. https://doi.org/10.1073/pnas.1104295108
  5. Lee AA, Kim NH, Alvarez S, Ren H, DeGrandchamp JB, Lew LJN, Groves JT. Bimodality in Ras signaling originates from processivity of the Ras activator SOS without deterministic bistability. Science Advances 10(25):eadi0707, 2024. DOI: 10.1126/sciadv.adi0707. https://doi.org/10.1126/sciadv.adi0707
  6. Christensen SM, Tu HL, Jun JE, Alvarez S, Triplet MG, Iwig JS, Yadav KK, Bar-Sagi D, Roose JP, Groves JT. One-way membrane trafficking of SOS in receptor-triggered Ras activation. Nature Structural & Molecular Biology 23(9):838-846, 2016. DOI: 10.1038/nsmb.3275. https://doi.org/10.1038/nsmb.3275
  7. McLaurin JD, Weiner OD. Multiple sources of signal amplification within the B-cell Ras/MAPK pathway. Molecular Biology of the Cell 30(13):1610-1620, 2019. DOI: 10.1091/mbc.e18-09-0560. https://doi.org/10.1091/mbc.e18-09-0560
  8. Bandaru P, Kondo Y, Kuriyan J. The interdependent activation of Son-of-Sevenless and Ras. Cold Spring Harbor Perspectives in Medicine 9(2):a031534, 2019. DOI: 10.1101/cshperspect.a031534. https://doi.org/10.1101/cshperspect.a031534
  9. McAffee DB, O'Dair MK, Lin JJ, Low-Nam ST, Wilhelm KB, Kim S, Morita S, Groves JT. Discrete LAT condensates encode antigen information from single pMHC:TCR binding events. Nature Communications 13:7446, 2022. DOI: 10.1038/s41467-022-35093-9. https://doi.org/10.1038/s41467-022-35093-9
  10. Lin CW, Nocka LM, Stinger BL, DeGrandchamp JB, Lew LJN, Alvarez S, Phan HT, Kondo Y, Kuriyan J, Groves JT. A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane. Proceedings of the National Academy of Sciences USA 119(19):e2122531119, 2022. DOI: 10.1073/pnas.2122531119. https://doi.org/10.1073/pnas.2122531119
  11. Hoang HM, Umutesi HG, Heo J. Allosteric autoactivation of SOS and its kinetic mechanism. Small GTPases 12(1):44-59, 2021. DOI: 10.1080/21541248.2019.1601954. https://doi.org/10.1080/21541248.2019.1601954
  12. Clark CE, Hasan M, Bousso P. A role for the immediate early gene product c-fos in imprinting T cells with short-term memory for signal summation. PLoS ONE 6(4):e18916, 2011. DOI: 10.1371/journal.pone.0018916. https://doi.org/10.1371/journal.pone.0018916
  13. Marangoni F, Murooka TT, Manzo T, Kim EY, Carrizosa E, Elpek NM, Mempel TR. The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells. Immunity 38(2):237-249, 2013. DOI: 10.1016/j.immuni.2012.09.012. https://doi.org/10.1016/j.immuni.2012.09.012
  14. Prasad A, Zikherman J, Das J, Roose JP, Weiss A, Chakraborty AK. Origin of the sharp boundary that discriminates positive and negative selection of thymocytes. Proceedings of the National Academy of Sciences USA 106(2):528-533, 2009. DOI: 10.1073/pnas.0805981105. https://doi.org/10.1073/pnas.0805981105
  15. Funasaki S, Hatano A, Nakatsumi H, Koga D, Sugahara O, Yumimoto K, Baba M, Matsumoto M, Nakayama KI. A stepwise and digital pattern of RSK phosphorylation determines the outcome of thymic selection. iScience 26(9):107552, 2023. DOI: 10.1016/j.isci.2023.107552. https://doi.org/10.1016/j.isci.2023.107552
  16. Huang J, Brameshuber M, Zeng X, Xie J, Li QJ, Chien YH, Valitutti S, Davis MM. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4+ T cells. Immunity 39(5):846-857, 2013. DOI: 10.1016/j.immuni.2013.08.036. https://doi.org/10.1016/j.immuni.2013.08.036
  17. Tkach KE, Barik D, Voisinne G, Malandro N, Hathorn MM, Cotari JW, Vogel R, Merghoub T, Wolchok J, Krichevsky O, Altan-Bonnet G. T cells translate individual, quantal activation into collective, analog cytokine responses via time-integrated feedbacks. eLife 3:e01944, 2014. DOI: 10.7554/eLife.01944. https://doi.org/10.7554/eLife.01944
  18. Tian T, Harding A, Inder K, Plowman S, Parton RG, Hancock JF. Plasma membrane nanoswitches generate high-fidelity Ras signal transduction. Nature Cell Biology 9(8):905-914, 2007. DOI: 10.1038/ncb1615. https://doi.org/10.1038/ncb1615
  19. Altan-Bonnet G, Germain RN. Modeling T cell antigen discrimination based on feedback control of digital ERK responses. PLoS Biology 3(11):e356, 2005. DOI: 10.1371/journal.pbio.0030356. https://doi.org/10.1371/journal.pbio.0030356
  20. Artyomov MN, Das J, Kardar M, Chakraborty AK. Purely stochastic binary decisions in cell signaling models without underlying deterministic bistabilities. Proceedings of the National Academy of Sciences USA 104(48):18958-18963, 2007. DOI: 10.1073/pnas.0706110104. https://doi.org/10.1073/pnas.0706110104