scholarly journals Development of Noonan syndrome by deregulation of allosteric SOS autoactivation

2020 ◽  
Vol 295 (39) ◽  
pp. 13651-13663 ◽  
Author(s):  
Hope Gloria Umutesi ◽  
Hanh My Hoang ◽  
Hope Elizabeth Johnson ◽  
Kwangho Nam ◽  
Jongyun Heo
Keyword(s):  
Noonan Syndrome ◽  
Genetic Disorder ◽  
Active Form ◽  
The Body ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Allosteric Site ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Ras Family

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants—V14I, T58I, and G60E—populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant–mediated development of Noonan syndrome.

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 R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

2007 ◽  
Vol 18 (5) ◽  
pp. 1850-1860 ◽  
Author(s):  
Akiyuki Takaya ◽  
Takahiro Kamio ◽  
Michitaka Masuda ◽  
Naoki Mochizuki ◽  
Hirofumi Sawa ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Recycling Endosomes ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Ras Family

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

Abstract P470: Disrupted HRAS/GRB2 Signaling In Induced-cardiomyocytes Derived From Patients With Noonan Syndrome Carrying SOS1 Gene Mutation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
VINOTH SIGAMANI ◽  
Narasimman Gurusamy ◽  
SHEEJA RAJASINGH ◽  
Rajasingh Johnson
Keyword(s):  
Gene Mutation ◽  
Noonan Syndrome ◽  
Guanine Nucleotide Exchange Factor ◽  
Signaling Molecules ◽  
Ras Signaling ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Background: Noonan syndrome (NS), a dominant autosomal genetic disorder that prevents normal development, and exhibits cardiac defects, which is estimated to appear in 50% to 90% of patients. Son of sevenless homolog 1 (SOS1) gene mutation has been identified as a major gene causing NS and attributes to the development of cardiomyopathy and congenital heart defects. SOS1 is a guanine nucleotide exchange factor for RAS and is known to interact with growth factor receptor-bound protein 2 (GRB2). Recently, we have generated induced pluripotent stem cells (iPSCs)-derived cardiomyocytes (iCMCs) from cardiac fibroblasts obtained from a NS patient carrying SOS1 gene variant 1654A>G. Hypothesis: Since NS is known to have aberrant RAS-MAPK signaling, we hypothesize that iCMCs derived from NS patient (NS-iCMCs) may have atypical RAS signaling leading to the development of cardiomyopathy. Methods and Results: We have compared the normal skin fibroblast-derived iPSCs (N-iPSCs) and N-iCMCs with NS patient-derived induced NS-iPSCs and NS-iCMCs. Our qRT-PCR results showed that the mRNA expressions of signaling molecules HRAS, GRB2 and SOS1 were significantly decreased in NS-iCMCs compared with N-iCMCs (Figure A), and further confirmed through the protein expression by Western immunoblotting (Figure B). These results were in association with a significantly decreased mRNA and protein expressions of cardiac transcription factor GATA4, and structural proteins alpha sarcomeric actinin-2 (ACTN2), cardiac troponin T (TNNT2) and tropomyosin alpha-1 (TPM1) in NS-iCMCs compared with N-iCMCs. Further studies are underway to explore the difference in the guanine nucleotide exchange factor (GEF) activity and ERK activation between NS-iCMCs and N-iCMCs. Conclusion: Our current findings clearly indicate that the SOS1-associated signaling molecules HRAS and GRB2 were disrupted in NS-iCMCs, which may result in the development of cardiomyopathy in NS patients.

scholarly journals C3G Protein, a New Player in Glioblastoma

2021 ◽  
Vol 22 (18) ◽  
pp. 10018
Author(s):  
Sara Manzano ◽  
Alvaro Gutierrez-Uzquiza ◽  
Paloma Bragado ◽  
Angel M Cuesta ◽  
Carmen Guerrero ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein A ◽  
Patient Treatment ◽  
Migration And Invasion ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Mesenchymal Phenotype ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Personalised Treatment

C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival.

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.

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 Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

2008 ◽  
Vol 181 (7) ◽  
pp. 1073-1081 ◽  
Author(s):  
Nadia Vadaie ◽  
Heather Dionne ◽  
Darowan S. Akajagbor ◽  
Seth R. Nickerson ◽  
Damian J. Krysan ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Active Form ◽  
Cell Polarization ◽  
Mitogen Activated Protein Kinase ◽  
Aspartyl Protease ◽  
Nucleotide Exchange ◽  
Mapk Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mitogen Activated Protein

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.

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