scholarly journals Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

2016 ◽  
Vol 113 (29) ◽  
pp. 8218-8223 ◽  
Author(s):  
William Y. C. Huang ◽  
Qingrong Yan ◽  
Wan-Chen Lin ◽  
Jean K. Chung ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Growth Factor Receptor ◽  
Cell Receptor ◽  
Nucleotide Exchange ◽  
Fast Kinetics ◽  
Surface Receptors ◽  
Signaling Systems ◽  
Downstream Signaling ◽  
Son Of Sevenless

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

scholarly journals Requirement for Ras Guanine Nucleotide Releasing Protein 3 in Coupling Phospholipase C-γ2 to Ras in B Cell Receptor Signaling

2003 ◽  
Vol 198 (12) ◽  
pp. 1841-1851 ◽  
Author(s):  
Masatsugu Oh-hora ◽  
Sachiko Johmura ◽  
Ari Hashimoto ◽  
Masaki Hikida ◽  
Tomohiro Kurosaki
Keyword(s):  
B Cell ◽  
Phospholipase C ◽  
Growth Factor Receptor ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Ras Activation ◽  
Attached Form

Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, we have generated Sos1/Sos2 double- or RasGRP3-deficient B cell lines and determined their ability to mediate Ras activation upon B cell receptor (BCR) stimulation. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-γ2–deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-γ2 regulates RasGRP3 localization and thereby Ras activation.

scholarly journals Vav Family Proteins Couple to Diverse Cell Surface Receptors

2000 ◽  
Vol 20 (17) ◽  
pp. 6364-6373 ◽  
Author(s):  
Sheri L. Moores ◽  
Laura M. Selfors ◽  
Jessica Fredericks ◽  
Timo Breit ◽  
Keiko Fujikawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Receptor Activation ◽  
Cell Surface Receptors ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Downstream Signaling

ABSTRACT Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.

scholarly journals Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events

2019 ◽  
Vol 12 (564) ◽  
pp. eaat8715 ◽  
Author(s):  
Jenny J. Y. Lin ◽  
Shalini T. Low-Nam ◽  
Katherine N. Alfieri ◽  
Darren B. McAffee ◽  
Nicole C. Fay ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cellular Response ◽  
Dwell Time ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Spatially Correlated ◽  
Order Of Magnitude

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long–dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

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 Conserved roles for receptor tyrosine kinase extracellular regions in regulating receptor and pathway activity

2020 ◽  
Vol 477 (21) ◽  
pp. 4207-4220
Author(s):  
Monica Gonzalez-Magaldi ◽  
Jacqueline M. McCabe ◽  
Haley N. Cartwright ◽  
Ningze Sun ◽  
Daniel J. Leahy
Keyword(s):  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Receptor Phosphorylation ◽  
Surface Receptors ◽  
Animal Development ◽  
Downstream Signaling ◽  
Extracellular Region ◽  
Downstream Effectors ◽  
Epidermal Growth ◽  
Pathway Inhibition

Receptor Tyrosine Kinases (RTKs) comprise a diverse group of cell-surface receptors that mediate key signaling events during animal development and are frequently activated in cancer. We show here that deletion of the extracellular regions of 10 RTKs representing 7 RTK classes or their substitution with the dimeric immunoglobulin Fc region results in constitutive receptor phosphorylation but fails to result in phosphorylation of downstream signaling effectors Erk or Akt. Conversely, substitution of RTK extracellular regions with the extracellular region of the Epidermal Growth Factor Receptor (EGFR) results in increases in effector phosphorylation in response to EGF. These results indicate that the activation signal generated by the EGFR extracellular region is capable of activating at least seven different RTK classes. Failure of phosphorylated Fc-RTK chimeras or RTKs with deleted extracellular regions to stimulate phosphorylation of downstream effectors indicates that either dimerization and receptor phosphorylation per se are insufficient to activate signaling or constitutive dimerization leads to pathway inhibition.

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.

scholarly journals Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Stimulates Tyrosine Kinase Cascades that Activate Ras and the Extracellular Signal Regulated Kinases (ERK1/2)

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3419-3427 ◽  
Author(s):  
Koji Shiraishi ◽  
Mario Ascoli
Keyword(s):  
Growth Factor ◽  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Classical Pathway ◽  
Nucleotide Exchange ◽  
Extracellular Signal ◽  
Son Of Sevenless ◽  
Negative Mutant ◽  
Formation Of Complexes

We show that activation of the recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of Shc (Src homology and collagen homology) and the formation of complexes containing Shc and Sos (Son of sevenless), a guanine nucleotide exchange factor for Ras. Because a dominant-negative mutant of Shc inhibits the LHR-mediated activation of Ras and the phosphorylation of ERK1/2, we conclude that the LHR-mediated phosphorylation of ERK1/2 is mediated, at least partially, by the classical pathway used by growth factor receptors. We also show that the endogenous epidermal growth factor receptor (EGFR) present in MA-10 cells is phosphorylated upon activation of the LHR. The LHR-mediated phosphorylation of the EGFR and Shc, the activation of Ras, and the phosphorylation of ERK1/2 are inhibited by expression of a dominant-negative mutant of Fyn, a member of the Src family kinases (SFKs) expressed in MA-10 cells and by PP2, a pharmacological inhibitor of the SFKs. These are also inhibited, but to a lesser extent, by AG1478, an inhibitor of the EGFR kinase. We conclude that the SFKs are responsible for the LHR-mediated phosphorylation of the EGFR and Shc, the formation of complexes containing Shc and Sos, the activation of Ras, and the phosphorylation of ERK1/2.

scholarly journals Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

Science ◽  
2014 ◽  
Vol 345 (6192) ◽  
pp. 50-54 ◽  
Author(s):  
Lars Iversen ◽  
Hsiung-Lin Tu ◽  
Wan-Chen Lin ◽  
Sune M. Christensen ◽  
Steven M. Abel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Specific Activity ◽  
Nucleotide Exchange ◽  
Turnover Rates ◽  
Highly Active ◽  
Stochastic Fluctuations ◽  
Ras Activation ◽  
Son Of Sevenless ◽  
Fluctuation Dynamics ◽  
Guanosine Triphosphatase

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

scholarly journals Sos-mediated activation of rac1 by p66shc

2006 ◽  
Vol 172 (6) ◽  
pp. 817-822 ◽  
Author(s):  
Firdous A. Khanday ◽  
Lakshmi Santhanam ◽  
Kenji Kasuno ◽  
Tohru Yamamori ◽  
Asma Naqvi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Nucleotide Exchange ◽  
Son Of Sevenless ◽  
Rac1 Gtpase ◽  
Src Homology ◽  
Ch2 Domain

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.

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