Combined HRAS and NRAS ablation induces a RASopathy phenotype in mice.

BACKGROUND: HRASKO/NRASKO double knockout mice exhibit exceedingly high rates of perinatal lethality due to respiratory failure caused by a significant lung maturation delay. The few animals that reach adulthood have a normal lifespan, but present areas of atelectasis mixed with patches of emphysema and normal tissue in the lung. METHODS: Eight double knockout and eight control mice were analyzed using micro-X-ray computerized tomography and a Small Animal Physiological Monitoring system. Tissues and samples from these mice were analyzed using standard histological and Molecular Biology methods and the significance of the results analyzed using a Student´s T-test. RESULTS: The very few double knockout mice surviving up to adulthood display clear craniofacial abnormalities reminiscent of those seen in RASopathy mouse models, as well as thrombocytopenia, bleeding anomalies, and reduced platelet activation induced by thrombin. These surviving mice also present heart and spleen hyperplasia, and elevated numbers of myeloid-derived suppressor cells in the spleen. Mechanistically, we observed that these phenotypic alterations are accompanied by increased KRAS-GTP levels in heart, platelets and primary mouse embryonic fibroblasts from these animals. CONCLUSIONS: Our data uncovers a new, previously unidentified mechanism capable of triggering a RASopathy phenotype in mice as a result of the combined removal of HRAS and NRAS.

Ras-GRF2 regulates nestin-positive stem cell density and onset of differentiation during adult neurogenesis in the mouse dentate gyrus.

Various parameters of neurogenesis were analyzed in parallel in the two neurogenic areas (the Dentate Gyrus[DG] and the Subventricular Zone[SVZ]/Rostral Migratory Stream[RMS]/Main Olfactory Bulb[MOB] neurogenic system) of adult WT and KO mouse strains for the Ras-GRF1/2 genes (Ras-GRF1-KO, Ras-GRF2-KO, Ras-GRF1/2-DKO). Significantly reduced numbers of doublecortin[DCX]-positive cells were specifically observed in the DG, but not the SVZ/RMS/MOB neurogenic region, of Ras-GRF2-KO and Ras-GRF1/2-DKO mice indicating that this novel Ras-GRF2-dependent phenotype is spatially restricted to a specific neurogenic area. Consistent with a role of CREB as mediator of Ras-GRF2 function in neurogenesis, the density of p-CREB-positive cells was also specifically reduced in all neurogenic regions of Ras-GRF2-KO and DKO mice. Similar levels of early neurogenic proliferation markers (Ki67, BrdU) were observed in all different Ras-GRF genotypes analyzed but significantly elevated levels of nestin-immunolabel, particularly of undifferentiated, highly ramified, A-type nestin-positive neurons were specifically detected in the DG but not the SVZ/RMS/MOB of Ras-GRF2-KO and DKO mice. Together with assays of other neurogenic markers (GFAP, Sox2, Tuj1, NeuN), these observations suggest that the deficit of DCX/p-CREB-positive cells in the DG of Ras-GRF2-depleted mice does not involve impaired neuronal proliferation but rather delayed transition from the stem cell stage to the differentiation stages of the neurogenic process. This model is also supported by functional analyses of DG-derived neurosphere cultures and transcriptional characterization of the neurogenic areas of mice of all relevant Ras-GRF genotypes suggesting that the neurogenic role of Ras-GRF2 is exerted in a cell-autonomous manner through a specific transcriptional program.

A translational systems biology approach in both animals and humans identifies a functionally related module of accumbal genes involved in the regulation of reward processing and binge drinking in males.

BACKGROUND: The mesolimbic dopamine system, composed primarily of dopaminergic neurons in the ventral tegmental area that project to striatal structures, is considered to be the key mediator of reinforcement-related mechanisms in the brain. Prompted by a genome-wide association meta-analysis implicating the Ras-specific guanine nucleotide-releasing factor 2 (RASGRF2) gene in the regulation of alcohol intake in men, we have recently shown that male Rasgrf2(-/-) mice exhibit reduced ethanol intake and preference accompanied by a perturbed mesolimbic dopamine system. We therefore propose that these mice represent a valid model to further elucidate the precise genes and mechanisms regulating mesolimbic dopamine functioning. METHODS: Transcriptomic data from the nucleus accumbens (NAcc) of male Rasgrf2(-/-) mice and wild-type controls were analyzed by weighted gene coexpression network analysis (WGCNA). We performed follow-up genetic association tests in humans using a sample of male adolescents from the IMAGEN study characterized for binge drinking (n = 905) and ventral striatal activation during an fMRI reward task (n = 608). RESULTS: The WGCNA analyses using accumbal transcriptomic data revealed 37 distinct "modules," or functionally related groups of genes. Two of these modules were significantly associated with Rasgrf2 knockout status: M5 (p < 0.001) and M6 (p < 0.001). In follow-up translational analyses we found that human orthologues for the M5 module were significantly (p < 0.01) enriched with genetic association signals for binge drinking in male adolescents. Furthermore, the most significant locus, originating from the EH-domain containing 4 (EHD4) gene (p < 0.001), was also significantly associated with altered ventral striatal activity in male adolescents performing an fMRI reward task (pempirical < 0.001). LIMITATIONS: It was not possible to determine the extent to which the M5 module was dysregulated in Rasgrf2(-/-) mice by perturbed mesolimbic dopamine signalling or by the loss of Rasgrf2 function in the NAcc. CONCLUSION: Taken together, our findings indicate that the accumbal M5 module, initially identified as being dysregulated in male Rasgrf2(-/-) mice, is also relevant for human alcohol-related phenotypes potentially through the modulation of reinforcement mechanisms in the NAcc. We therefore propose that the genes comprising this module represent important candidates for further elucidation within the context of alcohol-related phenotypes.

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release.

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca(2+)-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2(-/-) mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2(-/-) mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the I(A) potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive-delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells.

BACKGROUND: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those "pancreatic" phenotypes. RESULTS: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins.Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice. CONCLUSIONS: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

Optimized Protocol for Isolation and Culture of Murine Neonatal Primary Lung Fibroblasts.

During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.

GRF2 Is Crucial for Cone Photoreceptor Viability and Ribbon Synapse Formation in the Mouse Retina.

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2KO and GRF2WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2KO and double-knockout GRF2KO/CDC42KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

The RasGrf family of mammalian guanine nucleotide exchange factors.

RasGrf1 and RasGrf2 are highly homologous mammalian guanine nucleotide exchange factors which are able to activate specific Ras or Rho GTPases. The RasGrf genes are preferentially expressed in the central nervous system, although specific expression of either locus may also occur elsewhere. RasGrf1 is a paternally-expressed, imprinted gene that is expressed only after birth. In contrast, RasGrf2 is not imprinted and shows a wider expression pattern. A variety of isoforms for both genes are also detectable in different cellular contexts. The RasGrf proteins exhibit modular structures composed by multiple domains including CDC25H and DHPH motifs responsible for promoting GDP/GTP exchange, respectively, on Ras or Rho GTPase targets. The various domains are essential to define their intrinsic exchanger activity and to modulate the specificity of their functional activity so as to connect different upstream signals to various downstream targets and cellular responses. Despite their homology, RasGrf1 and RasGrf2 display differing target specificities and non overlapping functional roles in a variety of signaling contexts related to cell growth and differentiation as well as neuronal excitability and response or synaptic plasticity. Whereas both RasGrfs are activatable by glutamate receptors, G-protein-coupled receptors or changes in intracellular calcium concentration, only RasGrf1 is reported to be activated by LPA, cAMP, or agonist-activated Trk and cannabinoid receptors. Analysis of various knockout mice strains has uncovered a specific functional contribution of RasGrf1 in processes of memory and learning, photoreception, control of post-natal growth and body size and pancreatic ß-cell function and glucose homeostasis. For RasGrf2, specific roles in lymphocyte proliferation, T-cell signaling responses and lymphomagenesis have been described.

Critical Requirement of SOS1 for Development of BCR/ABL-Driven Chronic Myelogenous Leukemia.

We showed previously that the ABL-mediated phosphorylation of SOS1 promotes RAC activation and contributes to BCR-ABL leukemogenesis, suggesting the relevant role of SOS1 in the pathogenesis of CML. To try and obtain direct experimental evidence of the specific mechanistic implication of SOS1 in CML development, here, we combined a murine model of CML driven by a p210BCR/ABL transgene with our tamoxifen-inducible SOS1/2-KO system in order to investigate the phenotypic impact of the direct genetic ablation of SOS1 or SOS2 on the pathogenesis of CML. Our observations showed that, in contrast to control animals expressing normal levels of SOS1 and SOS2 or to single SOS2-KO mice, p210BCR/ABL transgenic mice devoid of SOS1 presented significantly extended survival curves and also displayed an almost complete disappearance of the typical hematological alterations and splenomegaly constituting the hallmarks of CML. SOS1 ablation also resulted in a specific reduction in the proliferation and the total number of colony-forming units arising from the population of bone marrow stem/progenitor cells from p210BCR/ABL transgenic mice. The specific blockade of CML development caused by SOS1 ablation in p210BCR/ABL mice indicates that SOS1 is critically required for CML pathogenesis and supports the consideration of this cellular GEF as a novel, alternative bona fide therapeutic target for CML treatment in the clinic.

Ras GEF Mouse Models for the Analysis of Ras Biology and Signaling.

Animal models have become in recent years a crucial tool to understand the physiological and pathological roles of many cellular proteins. They allow analysis of the functional consequences of [1] complete or partial (time- or organ-limited) removal of specific proteins (knockout animals), [2] the exchange of a wild-type allele for a mutant or truncated version found in human illnesses (knock-in), or [3] the effect of overexpression of a given protein in the whole body or in specific organs (transgenic mice). In this regard, the study of phenotypes in Ras GEF animal models has allowed researchers to find specific functions for otherwise very similar proteins, uncovering their role in physiological contexts such as memory formation, lymphopoiesis, photoreception, or body homeostasis. In addition, mouse models have been used to unveil the functional role of Ras GEFs under pathological conditions, including Noonan syndrome, skin tumorigenesis, inflammatory diseases, diabetes, or ischemia among others. In the following sections, we will describe the methodological approaches employed for Ras GEF animal model analyses, as well as the main discoveries made.

The reversed intra- and extracellular pH in tumors as a unified strategy to chemotherapeutic delivery using targeted nanocarriers.

Solid tumors are complex entities, comprising a wide variety of malignancies with very different molecular alterations. Despite this, they share a set of characteristics known as "hallmarks of cancer" that can be used as common therapeutic targets. Thus, every tumor needs to change its metabolism in order to obtain the energy levels required for its high proliferative rates, and these adaptations lead to alterations in extra- and intracellular pH. These changes in pH are common to all solid tumors, and can be used either as therapeutic targets, blocking the cell proton transporters and reversing the pH changes, or as means to specifically deliver anticancer drugs. In this review we will describe how proton transport inhibitors in association with nanocarriers have been designed to block the pH changes that are needed for cancer cells to survive after their metabolic adaptations. We will also describe studies aiming to decrease intracellular pH in cancer using nanoparticles as molecular cages for protons which will be released upon UV or IR light exposure. Finally, we will comment on several studies that have used the extracellular pH in cancer for an enhanced cell internalization and tumor penetration of nanocarriers and a controlled drug delivery, describing how nanocarriers are being used to increase drug stability and specificity.

40 Years of RAS-A Historic Overview.

It has been over forty years since the isolation of the first human oncogene (HRAS), a crucial milestone in cancer research made possible through the combined efforts of a few selected research groups at the beginning of the 1980s. Those initial discoveries led to a quantitative leap in our understanding of cancer biology and set up the onset of the field of molecular oncology. The following four decades of RAS research have produced a huge pool of new knowledge about the RAS family of small GTPases, including how they regulate signaling pathways controlling many cellular physiological processes, or how oncogenic mutations trigger pathological conditions, including developmental syndromes or many cancer types. However, despite the extensive body of available basic knowledge, specific effective treatments for RAS-driven cancers are still lacking. Hopefully, recent advances involving the discovery of novel pockets on the RAS surface as well as highly specific small-molecule inhibitors able to block its interaction with effectors and/or activators may lead to the development of new, effective treatments for cancer. This review intends to provide a quick, summarized historical overview of the main milestones in RAS research spanning from the initial discovery of the viral RAS oncogenes in rodent tumors to the latest attempts at targeting RAS oncogenes in various human cancers.

Anthraquinones as Inhibitors of SOS RAS-GEF Activity.

Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers.

RasGRF1 disruption causes retinal photoreception defects and associated transcriptomic alterations.

RasGRF1 null mutant mice display impaired memory/learning and their hippocampus transcriptomic pattern includes a number of differentially expressed genes playing significant roles in sensory development and function. Odour avoidance and auditory brainstem response tests yielded normal results but electroretinographic analysis showed severe light perception impairment in the RasGRF1 knockouts. Whereas no structural alterations distinguished the retinas of wild-type and knockout mice, microarray transcriptional analysis identified at least 44 differentially expressed genes in the retinas of these Knockout animals. Among these, Crb1, Pttg1, Folh1 and Myo7a have been previously related to syndromes involving retina degeneration. Interestingly, over-expression of Folh1 would be expected to result in accumulation of its enzymatic product N-acetyl-aspartate, an event known to be linked to Canavan disease, a human cerebral degenerative syndrome often involving blindness and hearing loss. Consistently, in vivo brain nuclear magnetic resonance spectroscopy identified higher levels of N-acetyl-aspartate in our RasGRF1-/- mice and immunohistochemical analysis detected reduced levels of aspartoacylase, the enzyme which degrades N-acetyl-aspartate. These studies demonstrate for the first time the functional relevance of Ras signalling in mammalian photoreception and warrant further analysis of RasGRF1 Knockout mice as potential models to analyse molecular mechanisms underlying defective photoreception human diseases.

Concomitant deletion of HRAS and NRAS leads to pulmonary immaturity, respiratory failure and neonatal death in mice.

We reported previously that adult (HRAS-/-; NRAS-/-) double knockout (DKO) mice showed no obvious external phenotype although lower-than-expected numbers of weaned DKO animals were consistently tallied after crossing NRAS-KO and HRAS-KO mice kept on mixed genetic backgrounds. Using mouse strains kept on pure C57Bl/6 background, here we performed an extensive analysis of the offspring from crosses between HRAS-KO and NRAS-KO mice and uncovered the occurrence of very high rates of perinatal mortality of the resulting DKO littermates due to respiratory failure during the first postnatal 24-48 h. The lungs of newborn DKO mice showed normal organ structure and branching but displayed marked defects of maturation including much-reduced alveolar space with thick separating septa and significant alterations of differentiation of alveolar (AT1, AT2 pneumocytes) and bronchiolar (ciliated, Clara cells) cell lineages. We also observed the retention of significantly increased numbers of undifferentiated progenitor precursor cells in distal lung epithelia and the presence of substantial accumulations of periodic acid-Schiff-positive (PAS+) material and ceramide in the lung airways of newborn DKO mice. Interestingly, antenatal dexamethasone treatment partially mitigated the defective lung maturation phenotypes and extended the lifespan of the DKO animals up to 6 days, but was not sufficient to abrogate lethality in these mice. RNA microarray hybridization analyses of the lungs of dexamethasone-treated and untreated mice uncovered transcriptional changes pointing to functional and metabolic alterations that may be mechanistically relevant for the defective lung phenotypes observed in DKO mice. Our data suggest that delayed alveolar differentiation, altered sphingolipid metabolism and ceramide accumulation are primary contributors to the respiratory stress and neonatal lethality shown by DKO mice and uncover specific, critical roles of HRAS and NRAS for correct lung differentiation that are essential for neonatal survival and cannot be substituted by the remaining KRAS function in this organ.

The CSN3 subunit of the COP9 signalosome interacts with the HD region of Sos1 regulating stability of this GEF protein.

Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.

Spatial learning and long-term memory impairments in RasGrf1 KO, Pttg1 KO, and double KO mice.

BACKGROUND: RasGrf1 is a guanine-nucleotide releasing factor that enhances Ras activity. Human PTTG1 is an oncoprotein found in pituitary tumors and later identified as securin, a protein isolated from yeast with a reported role in chromosome separation. It has been suggested that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and their physiological response. At memory formation level, there are contradictory data regarding the role of RasGrf1, while Pttg1 has not been previously studied. Both proteins are expressed in the mammalian hippocampus, which is one of the key brain areas for spatial learning and memory. OBJECTIVE: The aim of this work was to study a potential link between RasGrf1 and Pttg1 in memory formation. METHOD: Spatial learning and memory test in the Pttg1 KO, RasGrf1 KO, and Pttg1-RasGrf1 double KO and their correspondent WT mice using a Barnes maze. RESULTS: In comparison with the WT control mice, Pttg1 KO mice learned how to solve the task in a less efficient way, suggesting problems in memory consolidation. RasGrf1 KO mice performance was similar to controls, and they learned to use the best searching strategy. Double KO mice reached a better spatial learning level than WT. CONCLUSION: A role for Pttg1 in memory consolidation/formation is suggested, while our RasGrf1 KO mice do not show hippocampus associated memory defects.

Targeted disruption of Ras-Grf2 shows its dispensability for mouse growth and development.

The mammalian Grf1 and Grf2 proteins are Ras guanine nucleotide exchange factors (GEFs) sharing a high degree of structural homology, as well as an elevated expression level in central nervous system tissues. Such similarities raise questions concerning the specificity and/or redundancy at the functional level between the two Grf proteins. grf1-null mutant mice have been recently described which showed phenotypic growth reduction and long-term memory loss. To gain insight into the in vivo function of Grf2, we disrupted its catalytic CDC25-H domain by means of gene targeting. Breeding among grf2(+/-) animals gave rise to viable grf2(-/-) adult animals with a normal Mendelian pattern, suggesting that Grf2 is not essential for embryonic and adult mouse development. In contrast to Grf1-null mice, analysis of grf2(-/-) litters showed similar size and weight as their heterozygous or wild-type grf2 counterparts. Furthermore, adult grf2(-/-) animals reached sexual maturity at the same age as their wild-type littermates and showed similar fertility levels. No specific pathology was observed in adult Grf2-null animals, and histopathological studies showed no observable differences between null mutant and wild-type Grf2 mice. These results indicate that grf2 is dispensable for mouse growth, development, and fertility. Furthermore, analysis of double grf1/grf2 null animals did not show any observable phenotypic difference with single grf1(-/-) animals, further indicating a lack of functional overlapping between the two otherwise highly homologous Grf1 and Grf2 proteins.