A Sos proteomimetic as a pan-Ras inhibitor.

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

Time-Series Chlorophyll Fluorescence Imaging Reveals Dynamic Photosynthetic Fingerprints of sos Mutants to Drought Stress.

Resistance to drought stress is one of the most favorable traits in breeding programs yet drought stress is one of the most poorly addressed biological processes for both phenomics and genetics. In this study, we investigated the potential of using a time-series chlorophyll fluorescence (ChlF) analysis to dissect the ChlF fingerprints of salt overly sensitive (SOS) mutants under drought stress. Principle component analysis (PCA) was used to identify a shifting pattern of different genotypes including sos mutants and wild type (WT) Col-0. A time-series deep-learning algorithm, sparse auto encoders (SAEs) neural network, was applied to extract time-series ChlF features which were used in four classification models including linear discriminant analysis (LDA), k-nearest neighbor classifier (KNN), Gaussian naive Bayes (NB) and support vector machine (SVM). The results showed that the discrimination accuracy of sos mutants SOS1-1, SOS2-3, and wild type Col-0 reached 95% with LDA classification model. Sequential forward selection (SFS) algorithm was used to obtain ChlF fingerprints of the shifting pattern, which could address the response of sos mutants and Col-0 to drought stress over time. Parameters including QY, NPQ and Fm, etc. were significantly different between sos mutants and WT. This research proved the potential of ChlF imaging for gene function analysis and the study of drought stress using ChlF in a time-series manner.

Lycopene Triggers Nrf2-AMPK Cross Talk to Alleviate Atrazine-Induced Nephrotoxicity in Mice.

Atrazine (ATR), an environmental persistent and bioaccumulative herbicide, has been associated with environmental nephrosis. Lycopene (LYC) exhibits important properties of nephroprotection, but there are limited data on the specific underlying mechanism. The primary objective of this study was to explore the therapeutic effect of LYC on ATR-induced nephrotoxicity in mice. The mice were divided randomly into 6 groups and treated as follows: control group (C), 5 mg/kg LYC group (L), 50 mg/kg ATR group (A1), 200 mg/kg ATR group (A2), 50 mg/kg ATR plus 5 mg/kg LYC group (A1+L), and 200 mg/kg ATR plus 5 mg/kg LYC group (A2+L) by oral gavage administration for 21 days. We found that pretreatment with LYC significantly suppressed the ATR-induced renal tubular epithelial cell swelling. Furthermore, LYC mitigated ATR-induced dysregulation of oxidative stress markers by reducing MDA, H2O2 levels, and increasing SOD, GPx, CAT concentration, and Nrf2 activation. Moreover, LYC activated the autophagic flux by a detectable change in autophagy-related genes (Beclin-1 and ATGs) and proteins (p62/SQSTM) and by the formation of autophagic vacuole (AV) and LC3 aggregation, in parallel with AMPK activation (pAMPK/AMPK). Herein, ATR-up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes, including quinoneoxidoreductase-1 (NQO1) and heme oxidase-1 (HO1), whereas LYC down-regulated those of the above genes. In addition, LYC suppressed ATR-induced activation of autophagy (increased LC3II/LC3I, ATGs, Beclin1, and p62, in parallel with increased AMPK activation). Collectively, our findings identified a cross talk between AMPK-activated autophagy and the Nrf2 signaling pathway in LYC-mediated nephroprotection against ATR-induced toxicity in mice kidney.

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

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 (15)N-labeled Ras as well as [(13)C]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.

Inhibition of CD40-induced N-Ras activation reduces leishmania major infection.

Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major-altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major-expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras-Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major-enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform-targeted antileishmanial immunotherapy and immunoprophylaxis.

DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris.

Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional up-regulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. Here we show that the signaling molecules downstream of receptor kinase (DRK) and daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

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.

Functional evaluation of circulating hematopoietic progenitors in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis>1,000/µl. Ten out of the 27 NS patients showed monocytosis>1,000/µl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/µl; range, 44-232) with a low rate of apoptosis (median, 2.1%; range, 0.4-12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/µl, 205.7; range, 58-1374; median apoptotic rate, 1.4%; range, 0.2-2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/µl, 4.9; range, 1.3-17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in comparison with the controls (median, 8.6%; range, 0-27.7% vs. median, 17.6%; range, 2.8-49.6%), suggesting an increased CD34+ cell survival. The functional evaluation of circulating hematopoietic progenitors showed specific patterns in NS and NS/MPD. These tests are a reliable integrative tool that, together with clinical data and other hematological parameters, could help detect NS patients with a high risk for a myeloproliferative evolution.

The mechanism of butyrate-induced collagen biosynthesis in cultured fibroblasts.

The data showing that butyrate may play an important role in cellular metabolism led us to study its effect on collagen biosynthesis in cultured fibroblasts. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. Confluent human dermal fibroblasts were treated with millimolar concentrations of sodium butyrate (NaB) for 48 hours. It was found that butyrate induced collagen biosynthesis and prolidase activity. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of Sos protein and MAP-kinases (ERK1, ERK2). It was found that the MEK inhibitor decreased collagen biosynthesis and expression of MAP-kinases (ERK1, ERK2), while NaB counteracted the process. The data suggests that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.

Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF.

Rac1 has a crucial role in epidermal growth factor (EGF)-induced membrane ruffling, lamellipodial protrusion, and cell migration. Several guanine nucleotide exchange factors (GEFs) including Sos1, Sos2, Tiam1 and Vav2 have been shown to transduce the growth signal from the EGF receptor to Rac1. To clarify the role of each GEF, we time-lapse imaged the EGF-induced activity change of Rac1 in A431 cells transfected with siRNA targeting each Rac1 GEF. Because knockdown of these GEFs suppressed EGF-induced Rac1 activation only partially, we looked for another Rac1 GEF downstream of the EGF receptor and found that Asef, a Rac1-Cdc42 GEF bound to the tumor suppressor APC, also contributed to EGF-induced Rac1 activation. Intriguingly, EGF stimulation induced phosphorylation of Tyr94 within the APC-binding region of Asef in a manner dependent on Src-family tyrosine kinases. The suppression of EGF-induced Rac1 activation in siRNA-treated cells was restored by wild-type Asef, but not by the Tyr94Phe mutant of Asef. This observation strongly argues for the positive role of Tyr94 phosphorylation in EGF-induced Asef activation following the activation of Rac1.

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS.

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline.

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.

New class of Son-of-sevenless (Sos) alleles highlights the complexities of Sos function.

The guanine nucleotide exchange factor (GEF) Son-of-sevenless (Sos) encodes a complex multidomain protein best known for its role in activating the small GTPase RAS in response to receptor tyrosine kinase (RTK) stimulation. Much less well understood is SOS's role in modulating RAC activity via a separate GEF domain. In the course of a genetic modifier screen designed to investigate the complexities of RTK/RAS signal transduction, a complementation group of 11 alleles was isolated and mapped to the Sos locus. Molecular characterization of these alleles indicates that they specifically affect individual domains of the protein. One of these alleles, SosM98, which contains a single amino acid substitution in the RacGEF motif, functions as a dominant negative in vivo to downregulate RTK signaling. These alleles provide new tools for future investigations of SOS-mediated activation of both RAS and RAC and how these dual roles are coordinated and coregulated during development.

Genetic identification of effectors downstream of Neu (ErbB-2) autophosphorylation sites in a Drosophila model.

The ErbB-2/Neu receptor tyrosine kinase plays a causal role in tumorigenesis in mammals. Neu's carboxyl terminus contains five phosphorylated tyrosines that mediate transformation through interaction with cytoplasmic SH2 or PTB containing adaptor proteins. We show that Drosophila adaptors signal from individual phosphotyrosine sites of rat Neu. Activated Neu expression in the midline glia suppressed apoptosis, similar to that seen with activated Drosophila EGF-R expression. Expression in eye and wing tissues generated graded phenotypes suitable for dosage-sensitive modifier genetics. Suppression of ErbB-2/Neu-induced phenotypes in tissues haplosufficient for genes encoding adaptor protein or second messengers suggests that pTyr 1227(YD) signals require Shc, and that pTyr 1253 (YE) signalling does not employ Ras, but does require Raf function. Signalling from pTyr (YB) was affected by a haplosufficiency in drk (Grb-2), and in genes thought to function downstream of Grb-2: dab, sos, csw (Shp-2), and dos (Gab-1). These data demonstrate the power of Drosophila genetics to unmask the molecules that signal from oncogenic ErbB-2/Neu.

Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1.

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR-/- (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines.

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP. However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.

Calmodulin and son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS.

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.