Ras Family Proteins Research Articles

Erratum: The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

Erratum: The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

Ras-Mediated Signal Transduction and Virulence in Human Pathogenic Fungi.

Signal transduction pathways regulating growth and stress responses are areas of significant study in the effort to delineate pathogenic mechanisms of fungi. In-depth knowledge of signal transduction events deepens our understanding of how a fungal pathogen is able to sense changes in the environment and respond accordingly by modulation of gene expression and re-organization of cellular activities to optimize fitness. Members of the Ras protein family are important regulators of growth and differentiation in eukaryotic organisms, and have been the focus of numerous studies exploring fungal pathogenesis. Here, the current data regarding Ras signal transduction are reviewed for three major pathogenic fungi: Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus. Particular emphasis is placed on Ras-protein interactions during control of morphogenesis, stress response and virulence.

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.

Prenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP

Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308–6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects.

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central β2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.

Coating cells with cationic silica–magnetite nanocomposites for rapid purification of integral plasma membrane proteins

This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica-magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell-lysis-buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10-fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10(7) Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN-based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.

Reduced expression of SynGAP, a neuronal GTPase-activating protein, enhances capsaicin-induced peripheral sensitization

Synaptic GTPase-activating protein (SynGAP) is a neuronal-specific Ras/Rap-GAP that increases the hydrolysis rate of GTP to GDP, converting Ras/Rap from the active into the inactive form. The Ras protein family modulates a wide range of cellular pathways including those involved in sensitization of sensory neurons. Since GAPs regulate Ras activity, SynGAP might be an important regulator of peripheral sensitization and pain. Therefore, we evaluated excitability, stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP), and nociception from wild-type (WT) mice and those with a heterozygous mutation of the SynGAP gene (SynGAP(+/-)). Our results demonstrate that SynGAP is expressed in primary afferent sensory neurons and that the capsaicin-stimulated CGRP release from spinal cord slices was two-fold higher from SynGAP(+/-) mice than that observed from WT mouse tissue, consistent with an increase in expression of the capsaicin receptor, transient receptor potential cation channel subfamily V member 1 (TRPV1), in SynGAP(+/-) dorsal root ganglia. However, there was no difference between the two genotypes in potassium-stimulated release of CGRP, the number of action potentials generated by a ramp of depolarizing current, or mechanical hypernociception elicited by intraplantar injection of capsaicin. In contrast, capsaicin-induced thermal hypernociception occurred at lower doses of capsaicin and had a longer duration in SynGAP(+/-) mice than WT mice. These results provide the first evidence that SynGAP is an important regulator of neuropeptide release from primary sensory neurons and can modulate capsaicin-induced hypernociception, demonstrating the importance of GAP regulation in signaling pathways that play a role in peripheral sensitization.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-RasQ61L or K-RasG12D) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

Multiple Cellular Proteins Modulate the Dynamics of K-ras Association with the Plasma Membrane

Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDE δ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.

Chemotaxis: new role for Ras revealed

A recent study of chemotaxis revealed a new role for the proto-oncogene Ras in the social ameba Dictyostelium discoideum. Chemotaxis, the directional movement of cells toward chemokines and other chemoattractants, plays critical roles in diverse physiological processes, such as mobilization of immune cells to fight invading microorganisms, targeting of metastatic cancer cells to specific tissues, and guidance of sperm cells to ova during fertilization. This work, published in the July 26 issue of The Journal of Cell Biology, was conducted in Dr. Devreotes’ lab at John Hopkins University and Dr. Parent’s lab at National Cancer Institute. This research team demonstrated that RasC functions as an upstream regulator of TORC2 and thereby governs the effects of TORC2-PKB signaling on the cytoskeleton and cell migration. Many of the core components of the underlying chemotaxis signaling network have been elucidated in D. discoideum. Chemoattractants are sensed by G-protein-coupled receptors (GPCRs), which leads to the activation heterotrimeric Gproteins, small Ras-like G-proteins, and phosphoinsositide 3kinase (PI3K), resulting in the generation of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). This phospholipid, in turn, prompts the membrane translocation of proteins containing pleckstrin homology (PH) domains, such as cytosolic regulator of adenylyl cyclase (CRAC) and protein kinase B (PKB), which regulate the cytoskeleton rearrangements during chemotaxis. Importantly, many of these components and their activation are highly localized to the leading edge of cells undergoing chemotaxis, assuring that cytoskeletal changes needed for directional movement are spatially restricted (reviewed in Jin et al., 2009). Although it has been well established that the PIP3 pathway plays an important role in the regulation of chemotaxis, additional pathways that act in parallel with the PIP3 pathway have recently been revealed. For instance, phospholipase A2 (PLA2) was reported to mediate chemotaxis in parallel with PIP3 pathway (Chen et al., 2007). In addition, a PIP3independent pathway in which PKB is activated by TORC2 (target of rapamycin complex 2) was found to regulate chemotaxis (Lee et al., 2005; Kamimura et al., 2008). However, the mechanism by which TORC2 is regulated in chemotaxis was poorly understood prior to the publication of the recent report by Cai et al. (2010). D. discoideum possesses two PKB homologs, namely PKBA, which contains a PH domain and is dynamically recruited to the plasma membrane by PIP3, and PKBR1, which is tethered to the plasma membrane via N-terminal myristoylation. In their previous work, the authors discovered that both PKBA and PKBR1 are activated by TORC2mediated phosphorylation of their hydrophobic motifs (HMs) (Kamimura et al., 2008) and phosphoinositide-dependent kinase (PDK)-mediated phosphorylation of their activation loops (ALs) (Kamimura and Devreotes, 2010). In the present study, Cai and colleagues investigated whether Ras family proteins activate TORC2 and, if so, what are the effects of this Ras-TORC2 pathway on chemotactic responsiveness. In order to examine whether Ras proteins are required for TORC2-mediated activation of PKB, the scientists first determined the PKB activity in different Ras knock-out cells. They found that phosphorylation of the HM of PKBR1, the ALs of both PKBR1 and PKBA, and many PKB substrates were significantly reduced in rasC but not rasG cells relative to wild-type cells. To further explore rasC's role in PKB activation, the authors examined the consequences of expressing activated RasC (RasC) and found that it dramatically prolonged the phosphorylation kinetics of the PKBR1 and multiple PKB substrates, suggesting that RasC is indeed involved in regulating the PKB pathway. In addition, RasC expression also prolonged actin polymerization and impaired chemotaxis in wild-type cells, effects that were suppressed in cells lacking Pianissimo (piaA), an essential component of TORC2. Taken together these findings

Resistance to chemotherapeutic agents and promotion of transforming activity mediated by embryonic stem cell-expressed Ras (ERas) signal in neuroblastoma cells

Neuroblastoma is a common childhood tumor derived from neural crest precursor cells. In the present study, we investigated the expression and function of embryonic stem cell-expressed Ras (ERas), a novel Ras family protein previously reported as the specific expression gene in embryonic stem cells (ES cells), in neuroblastoma cell lines. Our results showed that the expressions of ERas were detected in neuroblastoma cell lines by RT-PCR and Western blotting. Therefore, we transfected a full length ERas expression vector into the neuroblastoma cell line SH-SY5Y, which has weak endogenous expression of ERas, and obtained clones with higher levels of expression. Overexpression of ERas did not increase the growth rate of the ERas transfectants but promoted their transforming activity. The ERas transfectants were more resistant to all the chemotherapy agents than the parental cell line. The ability of ERas to rescue cells from the toxic effect of chemotherapeutic agents was inhibited by the phosphatidylinositol 3'-kinase (PI3K) inhibitor PD294002. These results show that the ERas/PI3K pathway may provide resistance to chemotherapy and promote transforming activity in neuroblastoma.

Neurofibromatosis type 1: diagnosis and recent advances

Neurofibromatosis type 1 (NF1) is a familial tumour predisposition syndrome with no current treatment regime. Neurofibromin, the NF1 gene product, is a Ras-GAP protein that downregulates Ras and when inactivated leads to increased cell growth, proliferation and eventually tumorigenesis. The Ras family proteins have a central role in cell biology and are of prime importance in cancer development. This article reviews recent advances from the past 5 years, covering aspects of molecular and clinical diagnosis of NF1, genotype/phenotype studies, the RASopathies, NF1 tumorigenesis, mouse NF1 models and potential therapies. What reader will gain: The reader is introduced to recent advances in NF1 that focus on clinical and molecular diagnosis. The reader should gain a better understanding of the molecular basis of NF1 and of other NF1-like syndromes, and the challenges presented for clinical management. A molecular diagnosis can usually be made in most NF1 patients using a battery of gene screening techniques. The sensitivity and specificity of mutation detection will increase with the introduction of new technologies and new bioinformatic tools. Pathogenic somatic mutations of the NF1 gene are increasingly being identified in tumours not usually associated with NF1, a clear indication that neurofibromin has a much wider biological role and has an importance far beyond NF1.

Microdeletion at chromosome 4q21 defines a new emerging syndrome with marked growth restriction, mental retardation and absent or severely delayed speech

BackgroundGenome-wide screening of large patient cohorts with mental retardation using microarray-based comparative genomic hybridisation (array-CGH) has recently led to identification several novel microdeletion and microduplication syndromes.MethodsOwing to the national array-CGH...

Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

A Novel Proteomic Approach to Define Leukemia Cell Resistance to Farnesyltransferase Inhibitors.

Abstract 3761Poster Board III-697Farnesylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as RAS. Methods allowing rapid and selective detection of protein farnesylation are fundamental for the understanding of farnesylated protein function and for monitoring efficacy of farnesyltransferase inhibitors (FTI). While the natural substrates for prenyltransferases are farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), FTase has been shown to incorporate isoprenoid analogues into protein substrates. Materials and MethodsFTase targets in three different myeloid leukemia cell lines (HL-60, K562 and NB-4) were labeled using the unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP) in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect AG-modified proteins. This highly effective labeling/detection method was coupled with two-dimensional electrophoresis (2-DE) and subsequent Western blotting. Identities of individual protein spots were determined by overlaying anti-AG immunoblots and immunoblots probed with antibodies specific for different known prenylation target proteins, such as RAS-family proteins. The clinically tested FTIs BMS-214,662 and L-778,123 as well as the nitrogen-containing bisphosphonate zoledronate were used to further validate the specificity of this labeling technique. As a proof of principle of the ability of this approach to identify target proteins important for FTI resistance, RNA interference (RNAi) was applied to investigate the role of one of the specific protein targets (K-RAS) which remained AG-labelled in the presence of FTI BMS-214,662. Following confirmation of K-RAS silencing by Western blotting, flow cytometric analysis of Annexin-V stained cells was used to quantify the effect of K-RAS silencing on BMS-214,662-induced apoptosis. ResultsMetabolic labeling of prenylated proteins with anilinogeranyl (AG) occurred in a time-dependent manner. This method provided increased resolution of the prenylated proteome as we were able to detect as many as ten distinct protein spots corresponding to a single band at approximately 20 kDa observed by 1D SDS-PAGE. Some of the proteins we have identified using this overlay method include H-RAS, K-RAS, N-RAS, Rap1, RhoB, RhoC, pre-Lamin A, Lamin B, and LKB1. AG-specific signals decreased upon FTI treatment, further substantiating the specificity of this method. Additional evidence of the specificity of this approach was the observation that the bisphosphonate inhibitor zoledronate did not inhibit protein AG-labeling. Interestingly, this method allowed direct evaluation of specific FTI targets as demonstrated by the different efficacies we observed between the two FTIs studied here – BMS-214,662 and L-778,123. This method even allowed identification of subtle differences among the leukemia cell lines tested. Closer evaluation of the farnesylated proteome demonstrated clear differences between BMS-214,662 and L-778,123, consistent with earlier reports that FTIs elicit numerous cellular effects, including induction of apoptosis and cytostatic effects. While BMS-214,662 effectively inhibited farnesylation of the majority of larger molecular weight proteins, L-778,123 also blocked prenylation of smaller molecular weight proteins such as N-RAS and K-RAS. However, RhoB and RhoC remained farnesylated regardless of FTI treatment. BMS-214,662-induced apoptosis was substantially potentiated by RNAi knock-down of K-RAS expression. ConclusionsThis snapshot approach allowed simple, rapid and dynamic analysis of the complex farnesylated proteome in leukemia cells. Our results demonstrate that this method can be used to identify and validate specific inhibitor targets. Importantly, this approach also successfully identified proteins (e.g. K-RAS) which may be important for resistance to some FTIs, and thus may be useful in directing development of more efficient therapeutic regimens. Disclosures:No relevant conflicts of interest to declare.

RasGEF1A and RasGEF1B are guanine nucleotide exchange factors that discriminate between Rap GTP‐binding proteins and mediate Rap2‐specific nucleotide exchange

The highly conserved RasGEF1 family of proteins contain a C-terminal CDC25-Ras exchange motif domain and an N-terminal RasGEF-N domain, and are of unknown function and specificity. Using purified RasGEF1A and RasGEF1B proteins, as well as Ras family proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. Although Rap2 was implicated in the regulation of cell adhesion, the establishment of cell morphology, and the modulation of synapses in neurons, no specific guanine nucleotide exchange factor for Rap2 was previously identified. Using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B.

Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.

Aspergillus fumigatusRasA Regulates Asexual Development and Cell Wall Integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.

Suppression of non-small cell lung tumor development by the let-7 microRNA family

Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that let-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-Ras(G12D)-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, let-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-Ras(G12D) largely rescued let-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden.

PLCε Selectively Transduces Thrombin Versus LPA Signals to Astrocyte Proliferation Through Rap1 and Rho

Phospholipase C epsilon (PLCε) is a recently discovered PLC that, in contrast to other PLCs, responds directly to inputs from Ras and Rho family small G‐proteins. Using primary astrocytes isolated from a PLCε‐deficient mouse strain we show that endogenous G‐protein coupled receptors for LPA, S1P and thrombin utilize predominantly PLCε for inositol phosphate (InsP) and DAG formation. LPA‐ and S1P‐mediated InsP production was sensitive to Pertussis toxin (65±8% and 74±9% inhibition respectively) whereas the thrombin response was reduced by C3 toxin (68±13% inhibition), suggesting that these ligands activate PLCε through different mechanisms, a Gi pathway for LPA and S1P and a Rho mediated pathway for thrombin. Thrombin stimulated [3H]thymidine incorporation mediated by was markedly reduced in KO astrocytes (66±2%) and further studies revealed that PLCε is required for thrombin‐induced sustained ERK phosphorylation. Activation of a Ras family protein, Rap1, was required for ERK phosphorylation and DNA synthesis and the activation of this small G‐protein was abolished in KO cells. Notably LPA‐induced ERK and mitogenic effects were not Rap1 or PLCε dependent. Thus PLCε is selectively utilized by thrombin vs. LPA to transduce mitogenic signals through mechanism distinct from its role in generation of PLC‐derived second messengers.National Institute of Health Grant GM 36927 and GM 53536.