Ral GTPases Research Articles

Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells

The small GTPase RalA has been implicated in glucose uptake in insulin-stimulated adipocytes, although it remains unclear whether RalA has a similar role in insulin signaling in other types of cells. Recently, we have demonstrated that the Rho family GTPase Rac1 has a critical role in insulin-dependent glucose uptake in myoblast culture and mouse skeletal muscle. However, the mechanisms underlying Rac1-dependent glucose uptake, mostly mediated by the plasma membrane translocation of the glucose transporter GLUT4, remain largely unknown. The purpose of this study is to examine the involvement of RalA in Rac1 regulation of the translocation of GLUT4 to the plasma membrane in muscle cells. Ectopic expression of a constitutively activated RalA mutant indeed stimulated GLUT4 translocation, suggesting an important role of RalA also in muscle cells. GLUT4 translocation induced by constitutively activated mutation of Rac1 or more physiologically by upstream Rac1 regulators, such as phosphoinositide 3 kinase and the guanine nucleotide exchange factor FLJ00068, was abrogated when the expression of RalA was downregulated by RNA interference. The expression of constitutively activated Rac1, on the other hand, caused GTP loading and subcellular redistribution of RalA. Collectively, we propose a novel mechanism involving RalA for Rac1-mediated GLUT4 translocation in skeletal muscle cells.

Transcriptional Signatures of Ral GTPase Are Associated with Aggressive Clinicopathologic Characteristics in Human Cancer

RalA and RalB are small GTPases that support malignant development and progression in experimental models of bladder, prostate, and squamous cancer. However, demonstration of their clinical relevance in human tumors remains lacking. Here, we developed tools to evaluate Ral protein expression, activation, and transcriptional output and evaluated their association with clinicopathologic parameters in common human tumor types. To evaluate the relevance of Ral activation and transcriptional output, we correlated RalA and RalB activation with the mutational status of key human bladder cancer genes. We also identified and evaluated a transcriptional signature of genes that correlates with depletion of RalA and RalB in vivo. The Ral transcriptional signature score, but not protein expression as evaluated by immunohistochemistry, predicted disease stage, progression to muscle invasion, and survival in human bladder cancers and metastatic and stem cell phenotypes in bladder cancer models. In prostate cancer, the Ral transcriptional signature score was associated with seminal vesicle invasion, androgen-independent progression, and reduced survival. In squamous cell carcinoma, this score was decreased in cancer tissues compared with normal mucosa, validating the experimental findings that Ral acts as a tumor suppressor in this tumor type. Together, our findings show the clinical relevance of Ral in human cancer and provide a rationale for the development of Ral-directed therapies.

The Epac-Rap1 Signaling Pathway Controls cAMP-mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells

Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca(2+) or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.

PGE2 promotes renal carcinoma cell invasion through activated RalA

Incidence of kidney cancer is on the rise, and a better understanding of molecular mechanisms involved in the cancer invasion and metastasis is required for the development of curative therapeutics. In this study, we report that the proinflammatory cytokine prostaglandin E2 (PGE2) induces the malignant SN12C, but not benign HK2 kidney cell invasion. The PGE2 increases SN12C cell invasion through a signal pathway that encompasses EP2 and EP4, Akt, small GTPase RalA and Ral GTP inactivator RGC2. The results support the idea that targeted interference of EP2/EP4 signal to RalA GTP may provide benefit to patients diagnosed with advanced kidney cancer.

Abstract 2138: Tumorigenic and migratory potential of Ral GTPases in non-small cell lung cancers

Abstract The Ral GTPase family, consisting of the Ral A and B isoforms contributes to tumor formation, invasion and metastasis, however their role in non-small cell lung cancer (NSCLC) is yet to be validated. Recently, we investigated the role of Ral GTPases in NSCLC by in vitro expression, colony formation and migration assay, in vivo tumor formation experiments, and analysis of microarray data from NSCLC patients. In vitro, Ral A and Ral B was expressed in all NSCLC cell lines studied (14) to varying levels. Increased expression of Ral A or Ral B was more frequent in Ras mutant cell lines (Ral A elevated in 2/8 cell lines with mutant KRas vs. 0/6 cell lines with WT KRas, Ral B elevated in 5/8 cell lines with mutant KRas vs. 2/6 WT KRas). Soft agar experiments with transient knockdown of Ral A, Ral B and Ral A+B in these cell lines demonstrated significant decrease (p<0.05) in colony formation. Dual knockdown of Ral A and B had a 2 to 3 fold decrease (p<0.05) in NSCLC cell motility in wound healing assays. In vivo, knockdown of Ral GTPases resulted in reduced subcutaneous tumor growth (p<0.05) in mice injected with the knockdown cells. Microarray data from NSCLC patients was analyzed to study the correlation between Ral A and B expression with overall patient survival. High Ral A expression was associated with poor patient survival in these microarray datasets (p<0.05). The above observations demonstrate that Ral GTPases play a role in lung cancer tumorigenesis and migration. In the Ras signaling cascade, Ral GTPases are downstream of Ras along with PI3K and MAPK signaling pathways. KRas mutations in NSCLC carry a poor patient prognosis. Based on our observations, we posit that Ral GTPases can be a valid target for drug development against KRas mutated lung cancers along with PI3K and MEK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2138. doi:1538-7445.AM2012-2138

Abstract 3257: Nodal signalling in choriocarcinoma cells

Abstract Choriocarcinoma is an aggressive cancer that arises when the invasion of placental trophoblast cells into the endometrium becomes dysregulated. Trophoblast invasion is a complex phenomenon, dependent on cues that drive trophoblast invasion, as well as factors to ensure that placental invasion does not extend beyond the myometrium. Nodal, a member of the TGF-β superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. However, Nodal's exact role in placental function is unclear. Nodal signals by activating the ALK4/7, ActRIIB and Cripto-1 receptor complex and emerging evidence suggests that its signalling strength can be controlled at the level of receptor endocytosis into signaling endosomes. Two key regulators of receptor endocytosis and cell invasion are the α-arrestins, multifunctional scaffolding proteins and Ral GTPases, members of the Ras superfamily. Ral can bind to ActRIIB, and its activity can be regulated by Ras, as well as by α-arrestins, in a Ras-independent manner. We thus hypothesized that Nodal promotes trophoblast invasion via a α-arrestin/Ral-mediated pathway. The specific objectives of my study are to (i) determine the effects of Nodal on trophoblast invasion, (ii) elucidate a role for α-arrestin / Ral signaling in regulating Nodal-induced trophoblast invasion. We found that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells using Matrigel-coated transwell chamber assays. Interestingly, we found that the JAR choriocarcinoma cells responded differently to Nodal in that while HTR-8SV/neo invasion returned to baseline levels, JAR choricocarcinoma cells continued to invade even at the higher concentrations of Nodal (100ng/mL & 500ng/mL). For this reason, we decided to look at potential differences in the regulation of Nodal-induced invasion between the non-malignant HTR-8SV/neo trophoblast and JAR choricoarcinoma cell lines. We found that the JAR choriocarcinoma cells differentially expressed Ral and α-arrestin proteins compared to the HTR-8SV/neo cells. Additonally, endogenous Ral and α-arrestins associate with the ALK4 and 7 receptors, and knockdown of these molecules impaired Nodal-induced invasion. Thus, our data reveal a novel function for Nodal in modulating trophoblast invasion via a α-arrestin/Ral signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3257. doi:1538-7445.AM2012-3257

Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

Suppressing tumorigenic inflammation

A paper uncovers a RAS–RAL GEF signalling pathway that activates AKT and RAL GTPases to promote survival and motility, and which is inhibited by the expression of the anti-inflammatory mediator TIPE2.

Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the bcr–abl gene. RalA, one of the Ras superfamily of small GTPases, is a downstream molecule of bcr–abl fusion protein in ras signaling pathway, but its role in CML is poorly understood. Here, we first detected RalA level in CML cells, which is highly expressed and distributed mainly in the cytoplasm and/or partially in endomembrane. Next, siRNA was used to deplete RalA expression for elucidating its function. The results showed that siRNA RalA effectively inhibited cell viability, induced apoptosis and enhanced sensitivity of arsenic trioxide (ATO), and there are some synergistic effects of anti-CML between RalA siRNA and ATO. Finally, we found that ATO also could downregulate protein level of bcr–abl in K562 and KCL-22. Our research provides evidence that RalA might also serve as linchpin modulators in leukemia, and combinatorial therapies of dual inhibition of bcr–abl and ras signaling pathways have a great potential in treatment of CML.

LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation

Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found that LST1 induces nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined that LST1 recruits the actin-crosslinking protein filamin to the plasma membrane and interacts with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes.

RalA and RalB differentially regulate development of epithelial tight junctions

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.

Ral GTPases Regulate Cell-Mediated Cytotoxicity in NK Cells

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.

Aurora Mitochondrialis Drives Fission during Mitosis

Mitochondria proliferate by growth and partition during every cell-division cycle. Recently, Kashatus etal. (2011) reported that Aurora A kinase regulates the small GTPase RalA to mediate mitochondrial fission. This work illuminates the molecular mechanism behind mitochondrial inheritance in mammals and extends the functional repertoire of a key mitotic regulator.

RALA and RALBP1 regulate mitochondrial fission at mitosis

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission1. Equal distribution of mitochondria to daughter cells during mitosis requires fission2. Mitotic mitochondrial fission depends upon both the relocalization of large GTPase Drp1 to the outer mitochondrial membrane and phosphorylation of S616 on Drp1 by the mitotic kinase cyclin B/Cdk12. We now report that these processes are mediated by the small Ras-like GTPase RalA and its effector RalBP1 (RLIP76/RLIP1/RIP1)3,4. Specifically, the mitotic kinase Aurora A phosphorylates S194 of RalA, relocalizing it to the mitochondria, where it concentrates RalBP1 and Drp1. Furthermore, RalBP1 associates with cyclin B/Cdk1 kinase activity to foster phosphorylation of Drp1 on S616. Disrupting either RalA or RalBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B/Cdk1 converge upon RalA and RalBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.

Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.

SH3BP1, an Exocyst-Associated RhoGAP, Inactivates Rac1 at the Front to Drive Cell Motility

The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.

Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex

Projection neurons migrate from the ventricular zone to the neocortical plate during mouse brain development. Their overall movement is radial, but they become multipolar and move non-radially in the intermediate zone. Here we show that Reelin, the Rap1 GTPase, and N-cadherin (NCad) are important for multipolar neurons to polarize their migration towards the cortical plate. Inhibition and rescue experiments indicate that Reelin regulates migration through Rap1 and Akt, and that Rap1-regulated GTPases, RalA/B, Rac1 and Cdc42, are also involved. We find that Rap1 regulates plasma membrane localization of N-cadherin, and N-cadherin rescues radial polarization when Rap1 is inhibited. Curiously, inhibition of Rap1 or N-cadherin has little effect on glia-dependent locomotion. We propose a multi-step mechanism in which Reelin activates Rap1, Rap1 up-regulates N-cadherin, and N-cadherin is needed to orient cell migration.

Abstract LB-16: Inhibition of RalA as a novel strategy for targeting ovarian cancer

Abstract Abstract: The Ral (Ras like) GTPase guanyl nucleotide-binding proteins, RalA and RalB are direct effectors of pro-oncogene Ras. RalA and RalB are implicated in tumorigenesis and play distinct roles in mediating carcinogenesis. The critical role of RalA in the initiation and progression of ovarian cancer is entirely unclear. Here, we report that RalA is overactivated in human ovarian cancer cells and human ovarian cancer tissues. The RalA downstream effectors (RalBP-1 and CDC42) and the regulators (GalGDS, PP2A Aα and β, as well as Aurora Kinase) that modulate RalA activation during the post-translational process were found to be de-regulated in ovarian cancer cells. ShRNA-mediated knockdown of RalA inhibited ovarian cancer cell proliferation and invasion. Geranyl-Geranyl transferase inhibitors (GGTI-2147) or Aurora Kinase Inhibitor (AKI) that inhibit RalA activation at post-translational levels suppressed ovarian cancer cell growth and invasion in-vitro. Additionally, subcutaneous (SC) mouse model with these two inhibitors also showed effective inhibition of tumor growth in-vivo. Therefore, RalA is a key player in the biology and dispersal of ovarian cancer cells and is a promising target for the development of novel therapeutics. To the best of our knowledge, the role of RalA in ovarian cancer tumorigenesis has not been documented before, therefore our strategy in targeting RalA for treatment of ovarian cancer merits highly in terms of novelty. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2011-LB-16

Control of Secondary Granule Release in Neutrophils by Ral GTPase

Neutrophil (polymorphonuclear leukocyte; PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocytosis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, we found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules but not primary or tertiary granules and caused dysregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, whereas chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Overexpression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, we found that Ral, which was associated with the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral translocation intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattractant stimulation.

Structural study of the Cdc25 domain from Ral-specific guanine-nucleotide exchange factor RalGPS1a

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.