scholarly journals A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS

Science ◽  
2019 ◽  
Vol 363 (6431) ◽  
pp. 1098-1103 ◽  
Author(s):  
William Y. C. Huang ◽  
Steven Alvarez ◽  
Yasushi Kondo ◽  
Young Kwang Lee ◽  
Jean K. Chung ◽  
...  
Keyword(s):  
Phase Transition ◽  
Single Molecule ◽  
Molecular Assembly ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Son Of Sevenless ◽  
Kinetic Proofreading

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-molecule assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS molecules on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (~50 seconds) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)–Grb2–SOS phosphotyrosine-driven phase transition at the membrane.

Relating cellular signaling timescales to single-molecule kinetics: A first-passage time analysis of Ras activation by SOS

2021 ◽  
Vol 118 (45) ◽  
pp. e2103598118
Author(s):  
William Y. C. Huang ◽  
Steven Alvarez ◽  
Yasushi Kondo ◽  
John Kuriyan ◽  
Jay T. Groves
Keyword(s):  
Single Molecule ◽  
Cellular Signaling ◽  
First Passage Time ◽  
Passage Time ◽  
Nucleotide Exchange ◽  
Time Analysis ◽  
First Passage ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Son Of Sevenless

Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor (GEF) that plays a central role in numerous cellular signaling pathways. Like many other signaling molecules, SOS is autoinhibited in the cytosol and activates only after recruitment to the membrane. The mean activation time of individual SOS molecules has recently been measured to be ∼60 s, which is unexpectedly long and seemingly contradictory with cellular signaling timescales, which have been measured to be as fast as several seconds. Here, we rectify this discrepancy using a first-passage time analysis to reconstruct the effective signaling timescale of multiple SOS molecules from their single-molecule activation kinetics. Along with corresponding experimental measurements, this analysis reveals how the functional response time, comprised of many slowly activating molecules, can become substantially faster than the average molecular kinetics. This consequence stems from the enzymatic processivity of SOS in a highly out-of-equilibrium reaction cycle during receptor triggering. Ultimately, rare, early activation events dominate the macroscopic reaction dynamics.

scholarly journals Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence

2015 ◽  
Vol 291 (4) ◽  
pp. 1703-1718 ◽  
Author(s):  
Uybach Vo ◽  
Navratna Vajpai ◽  
Liz Flavell ◽  
Romel Bobby ◽  
Alexander L. Breeze ◽  
...  
Keyword(s):  
Intrinsic Fluorescence ◽  
The Novel ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Site Specific ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Catalytic Sites ◽  
Son Of Sevenless

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

scholarly journals Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

2021 ◽  
Vol 118 (12) ◽  
pp. e2022403118
Author(s):  
Zahra Moghadamchargari ◽  
Mehdi Shirzadeh ◽  
Chang Liu ◽  
Samantha Schrecke ◽  
Charles Packianathan ◽  
...  
Keyword(s):  
Ion Mobility ◽  
Catalytic Domain ◽  
Therapeutic Interventions ◽  
Nucleotide Exchange ◽  
Molecular Assemblies ◽  
Oncogenic Ras ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Insight Into

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

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

2002 ◽  
Vol 156 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Metello Innocenti ◽  
Pierluigi Tenca ◽  
Emanuela Frittoli ◽  
Mario Faretta ◽  
Arianna Tocchetti ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Sequential Activation

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.

scholarly journals Direct Binding of the β1 Adrenergic Receptor to the Cyclic AMP-Dependent Guanine Nucleotide Exchange Factor CNrasGEF Leads to Ras Activation

2002 ◽  
Vol 22 (22) ◽  
pp. 7942-7952 ◽  
Author(s):  
Youngshil Pak ◽  
Nam Pham ◽  
Daniela Rotin
Keyword(s):  
Cyclic Amp ◽  
Adrenergic Receptor ◽  
Pdz Domain ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Activation

ABSTRACT G-protein-coupled receptors (GPCRs) can indirectly activate Ras primarily through the βγ subunits of G proteins, which recruit c-Src, phosphatidylinositol 3-kinase, and Grb2-SOS. However, a direct interaction between a Ras activator (guanine nucleotide exchange factor [GEF]) and GPCRs that leads to Ras activation has never been demonstrated. We report here a novel mechanism for a direct GPCR-mediated Ras activation. The β1 adrenergic receptor (β1-AR) binds to the PDZ domain of the cyclic AMP (cAMP)-dependent Ras exchange factor, CNrasGEF, via its C-terminal SkV motif. In cells heterologously expressing β1-AR and CNrasGEF, Ras is activated by the β1-AR agonist isoproterenol, and this activation is abolished in β1-AR mutants that cannot bind CNrasGEF or in CNrasGEF mutants lacking the catalytic CDC25 domain or cAMP-binding domain. Moreover, the activation is transduced via Gsα and not via Gβγ. In contrast to β1-AR, the β2-AR neither binds CNrasGEF nor activates Ras via CNrasGEF after agonist stimulation. These results suggest a model whereby the physical interaction between the β1-AR and CNrasGEF facilitates the transduction of Gsα-induced cAMP signal into the activation of Ras. The present study provides the first demonstration of direct physical association between a Ras activator and a GPCR, leading to agonist-induced Ras activation

scholarly journals Crystal structure of Sar1-GDP at 1.7 Å resolution and the role of the NH2 terminus in ER export

2001 ◽  
Vol 155 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Mingdong Huang ◽  
Jacques T. Weissman ◽  
Sophie Béraud-Dufour ◽  
Peng Luan ◽  
Chenqian Wang ◽  
...  
Keyword(s):  
Sequence Divergence ◽  
Guanine Nucleotide ◽  
Amphipathic Helix ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Copii Vesicle ◽  
Er Export

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-Å structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an α1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the α1' helix, mediates the initial steps in COPII coat assembly for export from the ER.

scholarly journals Hepatocyte Growth Factor-induced Ras Activation Requires ERM Proteins Linked to Both CD44v6 and F-Actin

2007 ◽  
Vol 18 (1) ◽  
pp. 76-83 ◽  
Author(s):  
Véronique Orian-Rousseau ◽  
Helen Morrison ◽  
Alexandra Matzke ◽  
Thor Kastilan ◽  
Giuseppina Pace ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Signaling Cascade ◽  
Nucleotide Exchange ◽  
Erm Proteins ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Hepatocyte Growth

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a “signalosome” complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a “responder” to signal transduction. Here, we show now that F-actin acts as an “inducer” that actively organizes the signaling cascade.

Sign in / Sign up

Export Citation Format

Share Document