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Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence. This work was funded by the Catalan Government—AGAUR (2017 SGR-569), Ministerio de Ciencia e Innovaciön (PID2019-104859GB-I00; RTI2018-094739-B-I00; PID2019-104734RB-I00), and by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC). T Cemeli (FPU13/06590), M.Guasch (FPU17/00229), R. Navaridas (FPU18/04480), and M. Ribes (TALENT-IRBLleida) were supported by a pre-doctoral fellowship from Ministerio de Educación, Cultura y Deportes, and from Diputació de Lleida.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time., Cell Reports, 40 (13), ISSN:2666-3864, ISSN:2211-1247

Small GTPases of the Rho and Ras families are important regulators of Schwann cell biology. The Ras-like GTPases RalA and RalB act downstream of Ras in malignant peripheral nerve sheath tumors. However, the physiological role of Ral proteins in Schwann cell development is unknown. Using transgenic mice with ablation of one or both Ral genes, we report that Ral GTPases are crucial for axonal radial sorting. While lack of only one Ral GTPase was dispensable for early peripheral nerve development, ablation of both RalA and RalB resulted in persistent radial sorting defects, associated with hallmarks of deficits in Schwann cell process formation and maintenance. In agreement, ex vivo–cultured Ral-deficient Schwann cells were impaired in process extension and the formation of lamellipodia. Our data indicate further that RalA contributes to Schwann cell process extensions through the exocyst complex, a known effector of Ral GTPases, consistent with an exocyst-mediated function of Ral GTPases in Schwann cells., The Journal of Cell Biology, 218 (7), ISSN:0021-9525, ISSN:1540-8140

Background Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

More than a hundred proteins comprise the RAS superfamily of small GTPases. This family can be divided into RAS, RHO, RAB, RAN, ARF, and RAD subfamilies, with each shown to play distinct roles in human cells in both health and disease. The RAS subfamily has a well-established role in human cancer with the three genes, HRAS, KRAS, and NRAS being the commonly mutated in tumors. These RAS mutations, most often functionally activating, are especially common in pancreatic, lung, and colorectal cancers. Efforts to inhibit RAS and related GTPases have produced inhibitors targeting the downstream effectors of RAS signaling, including inhibitors of the RAF-mitogen-activated protein kinase/extracellular signal-related kinase (ERK)-ERK kinase pathway and the phosphoinositide-3-kinase-AKT-mTOR kinase pathway. A third effector arm of RAS signaling, mediated by RAL (RAS like) has emerged in recent years as a critical driver of RAS oncogenic signaling and has not been targeted until recently. RAL belongs to the RAS branch of the RAS superfamily and shares a high structural similarity with RAS. In human cells, there are two genes, RALA and RALB, both of which have been shown to play roles in the proliferation, survival, and metastasis of a variety of human cancers, including lung, colon, pancreatic, prostate, skin, and bladder cancers. In this review, we summarize the latest knowledge of RAL in the context of human cancer and the recent advancements in the development of cancer therapeutics targeting RAL small GTPases.

RalA and RalB are involved in cell migration and membrane dynamics. This study finds that ablation of RalGTPases impairs nerve regeneration and alters Schwann cell process formation; conversely, activation of RalGTPases enhancea Schwann cell process formation, migration, and axon myelination., RalA and RalB are small GTPases that are involved in cell migration and membrane dynamics. We used transgenic mice in which one or both GTPases were genetically ablated to investigate the role of RalGTPases in the Schwann cell (SC) response to nerve injury and repair. RalGTPases were dispensable for SC function in the naive uninjured state. Ablation of both RalA and RalB (but not individually) in SCs resulted in impaired axon remyelination and target reinnervation following nerve injury, which resulted in slowed recovery of motor function. Ral GTPases were localized to the leading lamellipodia in SCs and were required for the formation and extension of both axial and radial processes of SCs. These effects were dependent on interaction with the exocyst complex and impacted on the rate of SC migration and myelination. Our results show that RalGTPases are required for efficient nerve repair by regulating SC process formation, migration, and myelination, therefore uncovering a novel role for these GTPases.

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Summary The exocyst is an evolutionarily conserved protein complex that regulates vesicular trafficking and scaffolds signal transduction. Key upstream components of the exocyst include monomeric RAL GTPases, which help mount cell-autonomous responses to trophic and immunogenic signals. Here, we present a quantitative proteomics-based characterization of dynamic and signal-dependent exocyst protein interactomes. Under viral infection, an Exo84 exocyst subcomplex assembles the immune kinase Protein Kinase R (PKR) together with the Hippo kinase Macrophage Stimulating 1 (MST1). PKR phosphorylates MST1 to activate Hippo signaling and inactivate Yes Associated Protein 1 (YAP1). By contrast, a Sec5 exocyst subcomplex recruits another immune kinase, TANK binding kinase 1 (TBK1), which interacted with and activated mammalian target of rapamycin (mTOR). RALB was necessary and sufficient for induction of Hippo and mTOR signaling through parallel exocyst subcomplex engagement, supporting the cellular response to virus infection and oncogenic signaling. This study highlights RALB-exocyst signaling subcomplexes as mechanisms for the integrated engagement of Hippo and mTOR signaling in cells challenged by viral pathogens or oncogenic signaling.

Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14 – 57) which were characterized by 1H-NMR, 13C-NMR and LC/MS-MS. These compounds were assessed for their effect on the in vitro anchorage independent growth of human lung cancer cell line H2122 and IC50 values calculated. Two of the more potent compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0 h post i.p. (50 mg/Kg) dose.

RalGDS is a guanine nucleotide exchange factor that promotes the active GTP-bound form of Ral GTPases, RalA and RalB. GTP-bound Ras has the capacity to activate Ral GTPases at least in part by binding to the C-terminal Ras-binding domain (RBD) of RalGDS and directing the protein to Ral GTPases in the plasma membrane. In many cases, activation of Ral proteins complements other Ras effector pathways to carry out a cell function, but in others it opposes them. Moreover, in many cases activation of Ral proteins contributes to the oncogenic potential of Ras. However, in some cell types Ral proteins suppresses tumor formation, suggesting oncogenic stimuli that function through Ras may need to suppress Ral activation in order to transform cells. In this paper, we demonstrate a potential biochemical mechanism for such phenomena by showing that c-Met receptors promote the tyrosine phosphorylation of RalGDS at Y752 in its RBD, which blocks the binding of Ras to RalGDS.

Cancer cells with stem-like phenotype are frequently proliferative and show high resistance to chemotherapeutic agents. Specific cell markers to identify the cancer stem cells and reverse the drugs resistance are urgent needs in clinic cancer treatment. To identify the potential role of integrin β3 in melanoma stem cells. Flow cytometry and immunofluorescence were performed to detect the expression levels of integrin β3 and integrin β3 related signal molecules. qRT-PCR and western blotting were used to detect the signaling pathways induced by integrin β3. Colony formation analysis and melanoma-bearing mice treatment by chemotherapeutic agents and integrin β3 inhibitors were used to detect the curative effects. We proved that integrin β3 could serve as a marker of stem-like cancer cells in melanoma, along with the acquired chemotherapeutic drugs resistance. Furthermore, we observed that the membrane–proximal complex of integrin β3 with KRAS and Galectin-3 on the surface of melanoma cancer cells could recruit the RalB, resulting in the activation of TBK1. The phosphorylated TBK1 facilitates the activation of NF-κB signaling pathway, leading to the stem-like phenotype and drug resistance development in melanoma. Herein, the combination of cilengitide, an integrin β3 inhibitor, and chemotherapeutic agents were capable of suppressing the tumor growth and reversing the drug resistance induced by integrin β3. These findings identified integrin β3 as a driver of melanoma stem-like cells with drug resistance and revealed an innovative strategy in clinic melanoma treatment.

Objective— RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. Approach and Results— This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet–leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. Conclusions— Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet–leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

The membrane-bound mucin MUC4 is a high molecular weight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-ras mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-ras in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the post-transcriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer., Romain Vasseur is a recipient of a doctoral fellowship of Lille2 University. Nicolas Skrypek is a recipient of a PhD fellowship from the Centre Hospitalier Régional et Universitaire (CHRU) de Lille/région Nord-Pas de Calais. Audrey Vincent is the recipient of a postdoctoral fellowship from the Fondation ARC and Région Nord-Pas de Calais. Isabelle Van Seuningen is the recipient of a “Contrat Hospitalier de Recherche Translationnelle”/CHRT 2010, AVIESAN. This work is supported by grants from la Ligue Nationale Contre le Cancer (Equipe Labellisée Ligue 2010, IVS), from SIRIC ONCOLille, Grant INCaDGOSInserm 6041 (IVS, NJ) and from “Contrat de Plan Etat Région” CPER Cancer 2007–2013 (IVS).

In Caenorhabditis elegans, the RAL-1 GTPase regulates multivesicular body formation and fusion with the plasma membrane, thereby promoting exosome secretion; in mammals, RalA and RalB are required for the secretion of exosome-like vesicles., Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.

The Ral guanosine triphosphatases (GTPases), RalA and RalB, are members of the Ras superfamily of small GTPases. Research on Ral GTPases and their functions over the past 25 years has revealed the essential involvement of these GTPases in unique and diverse cellular processes including exocyst-mediated exocytosis and related cellular activities. Moreover, it is increasingly appreciated that the aberrant activation of Ral GTPases is one of the major causes of human tumourigenesis induced by oncogenic Ras. Recent evidence suggests that Ral signalling pathways may be potential therapeutic targets for the treatment of human cancers. This review summarizes recent advance in the investigation of Ral GTPases.

Activating RAS mutations are common in human tumors. These mutations are often markers for resistance to therapy and subsequent poor prognosis. So far, targeting the RAF-MEK-ERK and PI3K-AKT signaling pathways downstream of RAS is the only promising approach in the treatment of cancer patients harboring RAS mutations. RAL GTPase, another downstream effector of RAS, is also considered as a therapeutic option for the treatment of RAS-mutant cancers. The RAL GTPase family comprises RALA and RALB, which can have either divergent or similar functions in different tumor models. Recent studies on non-small cell lung cancer (NSCLC) have showed that different RAS mutations selectively activate specific effector pathways. This observation requires broader validation in other tumor tissue types, but if true, will provide a new approach to the treatment of RAS-mutant cancer patients by targeting specific downstream RAS effectors according to the type of RAS mutation. It also suggests that RAL GTPase inhibition will be an important treatment strategy for tumors harboring RAS glycine to cysteine (G12C) or glycien to valine (G12V) mutations, which are commonly found in NSCLC and pancreatic cancer.

Acute myeloid leukemia (AML) is a devastating malignancy for which novel treatment approaches are desperately needed. Ras signaling is an attractive therapeutic target for AML because a large proportion of AMLs have mutations in NRAS, KRAS, or genes that activate Ras signaling, and key Ras effectors are activated in virtually all AML patient samples. This has inspired efforts to develop Ras-targeted treatment strategies for AML. Due to the inherent difficulty and disappointing efficacy of targeting Ras proteins directly, many have focused on inhibiting Ras effector pathways. Inhibiting the major oncogenic Ras effectors, the mitogen-activated protein kinase (MAPK) and/or phosphatidylinositiol-3-kinase (PI3K) pathways, has generally demonstrated modest efficacy for AML. While this may be in part related to functional redundancy between these pathways, it is now clear that other Ras effectors have key oncogenic roles. Specifically, the Ras-like (Ral) GTPases have emerged as critical mediators of Ras-driven transformation and AML cell survival. Our group recently uncovered a critical role for RALB signaling in leukemic cell survival and a potential mediator of relapse following Ras-targeted therapy in AML. Furthermore, we found that RALB signaling is hyperactivated in AML patient samples, and inhibiting RALB has potent anti-leukemic activity in preclinical AML models. While key questions remain regarding the importance of RALB signaling across the genetically diverse spectrum of AML, the specific mechanism(s) that promotes leukemic cell survival downstream of RALB, and how to pharmacologically target RALB signaling effectively - RALB has emerged as a critical Ras effector and potential therapeutic target for AML.

Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.

IntroductionThe placenta is arguably the most anatomically variable organ in mammals even though its primary function is conserved.MethodUsing RNA-Seq, we measured the expression profiles of 55 term placentas of 14 species of mammals representing all major eutherian superordinal clades and marsupials, and compared the evolution of expression across clades.ResultsWe identified a set of 115 core genes which is expressed (FPKM≥10) in all eutherian placentas, including genes with immune-modulating properties (ANXA2,ANXA1,S100A11,S100A10, andLGALS1), cell-cell interactions (LAMC1,LUM, andLGALS1), invasion (GRB2andRALB) and syncytialization (ANXA5andANXA1). We also identified multiple pre-eclampsia associated genes which are differentially expressed inHomo sapienswhen compared to the other 13 species. Multiple genes are significantly associated with placenta morphology, includingEREGandWNT5Awhich are both associated with placental shape.Discussion115 genes are important for the core functions of the placenta in all eutherian species analyzed. The molecular functions and pathways enriched in the core placenta align with the evolutionarily conserved functionality of the placenta.

Cell migration is central to many developmental, physiologic and pathological processes, including cancer progression. The Ral GTPases (RalA and RalB) which act down-stream the Ras oncogenes, are key players in the coordination between membrane trafficking and actin polymerization. A major direct effector of Ral, the exocyst complex, works in polarized exocytosis and is at the center of multiple protein-protein interactions that support cell migration by promoting protrusion formation, front-rear polarization, and extra-cellular matrix degradation. In this review we describe the recent advancements in deciphering the molecular mechanisms underlying this role of Ral via exocyst on cell migration. Among others, we will discuss the recently identified cross-talk between Ral and Rac1 pathways: exocyst binds to a negative regulator (the RacGAP SH3BP1) and to the major effector (the Wave Regulatory Complex, WRC) of Rac1, the master regulator of protrusions. Next challenge will be to better characterize the dynamics in space and in time of these molecular interplays, to better understand the pleiotropic functions of Ral in both normal and cancer cells.

Because the deubiquitinating enzyme USP33 is involved in several important cellular processes (β-adrenergic receptor recycling, centrosome amplification, RalB signaling, and cancer cell migration), its levels must be carefully regulated. Using quantitative mass spectrometry, we found that the intracellular level of USP33 is highly sensitive to the activity of p97. Knockdown or chemical inhibition of p97 causes robust accumulation of USP33 due to inhibition of its degradation. The p97 adaptor complex involved in this function is the Ufd1-Npl4 heterodimer. Furthermore, we identified HERC2, a HECT domain-containing E3 ligase, as being responsible for polyubiquitination of USP33. Inhibition of p97 causes accumulation of polyubiquitinated USP33, suggesting that p97 is required for postubiquitination processing. Thus, our study has identified several key molecules that control USP33 degradation within the ubiquitin-proteasome system.

Introduction: KRAS mutations are poor prognostic markers for patients with non–small-cell lung cancer (NSCLC). RALA and RALB GTPases lie downstream of RAS and are implicated in RAS-mediated tumorigenesis. However, their biological or prognostic role in the context of KRAS mutation in NSCLC is unclear. Methods: Using expression analysis of human tumors and a panel of cell lines coupled with functional in vivo and in vitro experiments, we evaluated the prognostic and functional importance of RAL in NSCLC and their relationship to KRAS expression and mutation. Results: Immunohistochemical (N = 189) and transcriptomic (N = 337) analyses of NSCLC patients revealed high RALA and RALB expression was associated with poor survival. In a panel of 14 human NSCLC cell lines, RALA and RALB had higher expression in KRAS mutant cell lines whereas RALA but not RALB activity was higher in KRAS mutant cell lines. Depletion of RAL paralogs identified cell lines that are dependent on RAL expression for proliferation and anchorage independent growth. Overall, growth of NSCLC cell lines that carry a glycine to cystine KRAS mutation were more sensitive to RAL depletion than those with wild-type KRAS. The use of gene expression and outcome data from 337 human tumors in RAL-KRAS interaction analysis revealed that KRAS and RAL paralog expression jointly impact patient prognosis. Conclusion: RAL GTPase expression carries important additional prognostic information to KRAS status in NSCLC patients. Simultaneously targeting RAL may provide a novel therapeutic approach in NSCLC patients harboring glycine to cystine KRAS mutations.

Recent work has demonstrated that autophagy, a phylogenetically conserved, lysosome-mediated pathway of protein degradation, is a key participant in pathological cardiac remodeling. One common feature of cell growth and autophagy is membrane biogenesis and processing. The exocyst, an octomeric protein complex involved in vesicle trafficking, is implicated in numerous cellular processes, yet its role in cardiomyocyte plasticity is unknown. Here, we set out to explore the role of small G protein-dependent control of exocyst function and membrane trafficking in stress-induced cardiomyocyte remodeling and autophagy. First, we tested in cultured neonatal rat cardiomyocytes (NRCMs) two isoforms of Ral (RalA, RalB) whose actions are mediated by the exocyst. In these experiments, mTOR inhibition in response to starvation or Torin1 was preserved despite RalA or RalB knockdown; however, activation of autophagy was suppressed only in NRCMs depleted of RalB, implicating RalB as being required for mTOR-dependent cardiomyocyte autophagy. To define further the role of RalB in cardiomyocyte autophagy, we analyzed hearts from mice lacking RalGDS (Ralgds(-/-)), a guanine exchange factor (GEF) for the Ral family of small GTPases. RalGDS-null hearts were similar to wild-type (WT) littermates in terms of ventricular structure, contractile performance, and gene expression. However, Ralgds(-/-) hearts manifested a blunted growth response (p

Idiopathic epilepsy is characterized by seizures without a clear etiology and is believed to have a strong genetic component but exhibits a complex inheritance pattern. Genetic factors seem to confer a low seizure threshold to susceptible individuals and thereby enhance epileptogenesis. However, the identity of susceptibility genes and the mechanisms regulating seizure threshold are still poorly understood. Here, we describe that reduced expression of RalBP1, a downstream effector of the small GTPases RalA and RalB, lowers the seizure threshold in mice. The intraperitoneal injection of the chemoconvulsant pentylenetetrazol induced more severe seizures in RalBP1 hypomorphic mice than in their wild-type littermates. The reduction of RalBP1 in the brain has no effect on neuronal excitability, but does decrease the inhibitory synaptic transmission onto CA1 pyramidal neurons. This impaired synaptic inhibition was associated with the loss of GABAergic interneurons in the CA1 subfield of the hippocampus. The present study identifies RalBP1 as a gene regulating the seizure threshold in mice and provides direct evidence for the role of RalBP1 in synaptic inhibition in vivo.

Summary Macroautophagy is a bulk degradation process characterised by the formation of double-membrane vesicles, called autophagosomes, which deliver cytoplasmic substrates for degradation in the lysosome. It has become increasingly clear that autophagy intersects with multiple steps of the endocytic and exocytic pathways, sharing many molecular players. A number of Rab and Arf GTPases that are involved in the regulation of the secretory and the endocytic membrane trafficking pathways, have been shown to play key roles in autophagy, adding a new level of complexity to its regulation. Studying the regulation of autophagy by small GTPases that are known to be involved in membrane trafficking is becoming a scientific hotspot and may provide answers to various crucial questions currently debated in the autophagy field, such as the origins of the autophagosomal membrane. Thus, this Commentary highlights the recent advances on the regulation of autophagy by membrane-trafficking small GTPases (Rab, Arf and RalB GTPases) and discusses their putative roles in the regulation of autophagosome formation, autophagosome-dependent exocytosis and autophagosome-lysosome fusion.

RalA and RalB induce p27 cytoplasmic mislocalization. Focusing on RalA, this study shows that this effect is induced via RalBP1 with a dependence on p27 Ser-10 phosphorylation, whereas PLD1 counteracts this effect in a Ser-10–independent manner. As a result, RalBP1 activation abrogates TGF-β–mediated growth arrest in epithelial cells., Constitutive activation or overactivation of Ras signaling pathways contributes to epithelial tumorigenesis in several ways, one of which is cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27Kip1 (p27). We previously showed that such an effect can be mediated by activation of the Ral-GEF pathway by oncogenic N-Ras. However, the mechanism(s) leading to p27 cytoplasmic accumulation downstream of activated Ral remained unknown. Here, we report a dual regulation of p27 cellular localization by Ral downstream pathways, based on opposing effects via the Ral effectors RalBP1 and phospholipase D1 (PLD1). Because RalA and RalB are equally effective in mislocalizing both murine and human p27, we focus on RalA and murine p27, which lacks the Thr-157 phosphorylation site of human p27. In experiments based on specific RalA and p27 mutants, complemented with short hairpin RNA–mediated knockdown of Ral downstream signaling components, we show that activation of RalBP1 induces cytoplasmic accumulation of p27 and that this event requires p27 Ser-10 phosphorylation by protein kinase B/Akt. Of note, activation of PLD1 counteracts this effect in a Ser-10–independent manner. The physiological relevance of the modulation of p27 localization by Ral is demonstrated by the ability of Ral-mediated activation of the RalBP1 pathway to abrogate transforming growth factor-β–mediated growth arrest in epithelial cells.

Geranylgeranyltransferase I inhibitors (GGTIs) are presently undergoing advanced preclinical studies and have been shown to disrupt oncogenic and tumor survival pathways, to inhibit anchorage-dependent and -independent growth, and to induce apoptosis. However, the geranylgeranylated proteins that are targeted by GGTIs to induce these effects are not known. Here we provide evidence that the Ras-like small GTPases RalA and RalB are exclusively geranylgeranylated and that inhibition of their geranylgeranylation mediates, at least in part, the effects of GGTIs on anchorage-dependent and -independent growth and tumor apoptosis. To this end, we have created the corresponding carboxyl-terminal mutants that are exclusively farnesylated and verified that they retain the subcellular localization and signaling activities of the wild-type geranylgeranylated proteins and that Ral GTPases do not undergo alternative prenylation in response to GGTI treatment. By expressing farnesylated, GGTI-resistant RalA and RalB in Cos7 cells and human pancreatic MiaPaCa2 cancer cells followed by GGTI-2417 treatment, we demonstrated that farnesylated RalB, but not RalA, confers resistance to the proapoptotic and anti-anchorage-dependent growth effects of GGTI-2417. Conversely, farnesylated RalA but not RalB expression renders MiaPaCa2 cells less sensitive to inhibition of anchorage-independent growth. Furthermore, farnesylated RalB, but not RalA, inhibits the ability of GGTI-2417 to suppress survivin and induce p27(Kip1) protein levels. We conclude that RalA and RalB are important, functionally distinct targets for GGTI-mediated tumor apoptosis and growth inhibition.

Mutationally activated K-Ras can utilize a multitude of downstream effector proteins to promote oncogenesis. While the Raf and phosphoinositol 3-kinase effector pathways are the best-studied and validated, recent studies have established the critical importance of Ral guanine nucleotide exchange factor (RalGEF) activation of the RalA and RalB small GTPases in cancer biology. Due to recent evidence that the RalGEF-Ral pathway is necessary for the tumorigenic and metastatic potential of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) tumor cells, we investigated whether or not Ral signaling was necessary for KRAS mutant colorectal cancer (CRC) tumor cell growth. As in PDAC, we found upregulated RalA and RalB activation in CRC tumor cell lines and tumors. Surprisingly we found antagonistic roles for RalA and RalB in the regulation of CRC tumor cell anchorage-independent growth. This observation contrasts with PDAC, where RalA but not RalB is necessary for PDAC tumor cell anchorage-independent growth. Our results emphasize cancer cell type differences in Ral function and hence the need for distinct Ral targeted therapeutic approaches in the treatment of CRC vs. PDAC.

The closely related GTPases RalA and RalB are required for assembly of tight junction gate, but not fence, function. These activities depend on direct binding to the exocyst complex. Whereas RalA–exocyst complexes are required for exocytosis of junction proteins, RalB–exocyst complexes are required for endocytosis of these components., Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference–mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical–basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.

Cyclin-dependent kinase 5 (CDK5), a neuronal kinase that functions in migration, has been found to be activated in some human cancers in which it has been implicated in promoting metastasis. In this study, we investigated the role of CDK5 in pancreatic cancers in which metastatic disease is most common at diagnosis. CDK5 was widely active in pancreatic cancer cells. Functional ablation significantly inhibited invasion, migration, and anchorage-independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in the profound inhibition of Ras signaling through its critical effectors RalA and RalB. Conversely, restoring Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Our findings identify CDK5 as a pharmacologically tractable target to degrade Ras signaling in pancreatic cancer. Cancer Res; 70(11); 4460–9. ©2010 AACR.

The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.

SummaryAnchorage dependence of cell growth is a key metastasis-suppression mechanism that is mediated by effects of integrins on growth signaling pathways [1]. The small GTPase RalA is activated in metastatic cancers through multiple mechanisms and specifically induces anchorage independence [2–4]. Loss of integrin-mediated adhesion triggers caveolin-dependent internalization of cholesterol- and sphingolipid-rich lipid raft microdomains to the recycling endosomes; these domains serve as platforms for many signaling pathways, and their clearance from the plasma membrane (PM) after cell detachment suppresses growth signaling [5, 6]. Conversely, readhesion triggers their return to the PM and restores growth signaling. Activation of Arf6 by integrins mediates exit of raft markers from the recycling endosomes but is not sufficient for return to the PM. We now show that RalA but not RalB mediates integrin-dependent membrane raft exocytosis through the exocyst complex. Constitutively active RalA restores membrane raft targeting to promote anchorage-independent growth signaling. Ras-transformed pancreatic cancer cells also show RalA-dependent constitutive PM raft targeting. These results identify RalA as a key determinant of integrin-dependent membrane raft trafficking and regulation of growth signaling. They therefore define a mechanism by which RalA regulates anchorage dependence and provide a new link between integrin signaling and cancer.

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.

The small G proteins RalA/B have a crucial function in the regulatory network that couples extracellular signals with appropriate cellular responses. RalA/B are an important component of the Ras signaling pathway and, in addition to their role in membrane trafficking, are implicated in the initiation and maintenance of tumorigenic transformation of human cells. RalA and RalB share 85% sequence identity and collaborate in supporting cancer cell proliferation but have markedly different effects. RalA is important in mediating proliferation, while depletion of RalB results in transformed cells undergoing apoptosis. Crystal structures of RalA in the free form and in complex with its effectors, Sec5 and Exo84, have been solved. Here we have determined the solution structure of free RalB bound to the GTP analogue GMPPNP to an RMSD of 0.6 A. We show that, while the overall architecture of RalB is very similar to the crystal structure of RalA, differences exist in the switch regions, which are sensitive to the bound nucleotide. Backbone 15N dynamics suggest that there are four regions of disorder in RalB: the P-loop, switch I, switch II, and the loop comprising residues 116-121, which has a single residue insertion compared to RalA. 31P NMR data and the structure of RalB.GMPPNP show that the switch regions predominantly adopt state 1 (Ras nomenclature) in the unbound form, which in Ras is not competent to bind effectors. In contrast, 31P NMR analysis of RalB.GTP reveals that conformations corresponding to states 1 and 2 are both sampled in solution and that addition of an effector protein only partially stabilizes state 2.

Although it has been established that the activation of GTPases by non-hydrolyzable GTP stimulates neurotransmitter release from many different secretory cell types, the underlying mechanisms remain unclear. In the present study we aimed to elucidate the functional role(s) for endogenous Ras-like protein A (RalA) and RalB GTPases in GTP-dependent exocytosis. For this purpose stable neuroendocrine pheochromocytoma 12 (PC12) cell lines were generated in which the expressions of both RalA and RalB were strongly downregulated. In these double knock-down cells GTP-dependent exocytosis was reduced severely and was restored after the expression of RalA or RalB was reintroduced by transfection. In contrast, Ca2+-dependent exocytosis and the docking of dense core vesicles analyzed by electron microscopy remained unchanged in the double knock-down cells. Furthermore, the transfected RalA and RalB appeared to be localized primarily on the dense core vesicles in undifferentiated and nerve growth factor-differentiated PC12 cells. Our results indicate that endogenous RalA and RalB function specifically as GTP sensors for the GTP-dependent exocytosis of dense core vesicles, but they are not required for the general secretory pathways, including tethering of vesicles to the plasma membrane and Ca2+-dependent exocytosis.

Background & Aims High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. Methods Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. Results Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes ( Acaa2 , Hbs1l , Ralgapa2 , and Ubr2 ) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. Conclusions In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase–activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.

UNLABELLED Fibroblast growth factor 19 (FGF19) is an important postprandial enterokine which regulates liver metabolism and hepatocyte proliferation. However, the precise mechanism by which FGF19 regulates these cellular effects is poorly understood. Given that mechanistic target of rapamycin complex 1 (mTORC1) regulates numerous postprandial adaptations, we investigated the potential role of mTORC1 in FGF19 action. We found that FGF19 activated mTORC1 in HepG2 and HuH7 human hepatoma cells, differentiated 3T3-L1 adipocytes and mouse liver. FGF19 activates the mTORC1-p70S6K and extracellular signal-regulated kinase (Erk)-p90RSK pathways independently to regulate S6 in an additive manner in hepatoma cells, but it uses mTORC1 as the primary pathway to regulate S6 in 3T3-L1 adipocytes. Thus, mTORC1 is a novel mediator of FGF19 signaling, which can act in parallel with Erk or function as the primary pathway to regulate S6. The FGF19-induced mTORC1 pathway requires amino acids for efficient signaling; thus, involvement of mTORC1 confers amino acid sensitivity to FGF19 signaling. Although Akt and Erk are known to activate mTORC1, we found that FGF19 signals to mTORC1 through a third recently identified mTORC1 regulator, Ras-like (Ral) protein. Pharmacological or genetic inhibition of RalA or RalB abolished FGF19-induced mTORC1 activation, demonstrating that Ral proteins are required for FGF19 to activate mTORC1. FGF19 induced metabolic gene expression, fatty acid oxidation, cell growth, and proliferation in HepG2 cells; and these effects were abolished by mTORC1 inhibition, demonstrating an essential role of mTORC1 in FGF19 action. CONCLUSION mTORC1 is a novel and essential mediator of FGF19 action on metabolic and mitogenic programs; thus, the involvement of mTORC1 in FGF19 signaling is an important factor to consider when targeting the pathway for cancer or diabetes therapy. (Hepatology 2016;64:1289-1301).

Summary Background The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. Results We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. Conclusions RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

SummaryThe monomeric RalGTPases, RalA and RalB are recognized as components of a regulatory framework supporting tumorigenic transformation. Specifically, RalB is required to suppress apoptotic checkpoint activation, the mechanistic basis of which is unknown. Reported effector proteins of RalB include the Sec5 component of the exocyst, an octameric protein complex implicated in tethering of vesicles to membranes. Surprisingly, we find that the RalB/Sec5 effector complex directly recruits and activates the atypical IκB kinase family member TBK1. In cancer cells, constitutive engagement of this pathway, via chronic RalB activation, restricts initiation of apoptotic programs typically engaged in the context of oncogenic stress. Although dispensable for survival in a nontumorigenic context, this pathway helps mount an innate immune response to virus exposure. These observations define the mechanistic contribution of RalGTPases to cancer cell survival and reveal the RalB/Sec5 effector complex as a component of TBK1-dependent innate immune signaling.

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

SummaryRalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

The Ras-like small G-proteins RalA and RalB have achieved some notoriety as components of one of a growing variety of candidate Ras effector pathways. Recent work has demonstrated that Ral GTPase activation is required to support both the initiation and maintenance of tumorigenic transformation of human cells. The mechanistic basis for this support remains to be defined. However, the discovery that the exocyst is a direct effector complex for activated Ral proteins suggests that mobilization of polarized exocytosis might be a basic component of the biological framework supporting tumorigenic progression.

SummaryTo investigate the role of signaling by the small GTPase Ral, we have generated mice deficient for RalGDS, a guanine nucleotide exchange factor that activates Ral. We show that RalGDS is dispensable for mouse development but plays a substantial role in Ras-induced oncogenesis. Lack of RalGDS results in reduced tumor incidence, size, and progression to malignancy in multistage skin carcinogenesis, and reduced transformation by Ras in tissue culture. RalGDS does not appear to participate in the regulation of cell proliferation, but instead controls survival of transformed cells. Experiments performed in cells isolated from skin tumors suggest that RalGDS mediates cell survival through the activation of the JNK/SAPK pathway. These studies identify RalGDS as a key component in Ras-dependent carcinogenesis in vivo.

In mammals, Ral (Ras-like) GTPases have been implicated in the regulation of several cellular key processes such as oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics. Here we provide, for the first time, molecular data on a Ral homologue from a parasitic helminth. We have cloned and characterized the complete cDNA molecule and the chromosomal locus encoding a novel GTP binding protein, EmRal, of the human parasite Echinococcus multilocularis. The encoded protein contained all highly conserved amino acid residues of the protein family at corresponding positions and shared significant sequence homologies with human RalA (53% identity) and RalB (54%). Upon heterologous expression of EmRal in Escherichia coli, the recombinant protein was able to bind GTP, thus indicating functionality of the Echinococcus factor. Using an in vitro prenylation assay, the purified protein was shown to be geranylgernylated, but not farnesylated, in both rabbit reticulocyte and Echinococcus cell extracts. The EmRal mRNA was found to be processed via trans-splicing and, using RT-PCR and virtual Northern blot experiments, expression of the factor could be demonstrated for the larval stages metacestode and protoscolex during an infection of the intermediate host. The data presented herein provide a solid basis for further investigations on Ras-Ral signaling mechanisms in Echinococcus.

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.

The sec6/8 complex or exocyst is an octameric protein complex that functions during cell polarization by regulating the site of exocytic vesicle docking to the plasma membrane, in concert with small GTP-binding proteins. The Sec5 subunit of the mammalian sec6/8 complex binds Ral in a GTP-dependent manner. Here we report the crystal structure of the complex between the Ral-binding domain of Sec5 and RalA bound to a non-hydrolyzable GTP analog (GppNHp) at 2.1 A resolution, providing the first structural insights into the mechanism and specificity of sec6/8 regulation. The Sec5 Ral-binding domain folds into an immunoglobulin-like beta-sandwich structure, which represents a novel fold for an effector of a GTP-binding protein. The interface between the two proteins involves a continuous antiparallel beta-sheet, similar to that found in other effector/G-protein complexes, such as Ras and Rap1A. Specific interactions unique to the RalA.Sec5 complex include Sec5 Thr11 and Arg27, and RalA Glu38, which we show are required for complex formation by isothermal titration calorimetry. Comparison of the structures of GppNHp- and GDP-bound RalA suggests a nucleotide-dependent switch mechanism for Sec5 binding.

The RAS family of small GTPases play critical roles in many types of human cancer. Activating RAS mutations are the most frequent type of oncogene mutations in human cancers, and are especially common in pancreatic, lung, and colorectal cancer. However, failure to obtain clinically useful inhibitors for RAS or any other GTPases suggests this target family is a therapeutic challenge. Consequently, significant efforts have been shifted toward targeting of downstream effectors of RAS including the Raf-MEK-ERK kinase pathway and the PI3K-AKT-mTOR kinase pathway. A third arm of RAS effector signaling, RAL (Ras-like) has not been targeted until recently and like RAS, is activated, but unlike RAS is not commonly mutated in cancer. RAL also shares a high structure similarity with RAS and other GTPase of the RAS superfamily. The two isoforms RALA and RALB are both important drivers of the proliferation, survival and metastasis of multiple human cancers. In our recent publication we reported the discovery of small molecule inhibitors of RAL.1

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Delivery of cytoplasmic vesicles to discrete plasma-membrane domains is critical for establishing and maintaining cell polarity, neurite differentiation and regulated exocytosis. The exocyst is a multisubunit complex required for vectorial targeting of a subset of secretory vesicles. Mechanisms that regulate the activity of this complex in mammals are unknown. Here we show that Sec5, an integral component of the exocyst, is a direct target for activated Ral GTPases. Ral GTPases regulate targeting of basolateral proteins in epithelial cells, secretagogue-dependent exocytosis in neuroendocrine cells and assembly of exocyst complexes. These observations define Ral GTPases as critical regulators of vesicle trafficking.