Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells

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.     

The RalB small GTPase mediates formation of invadopodia through a GTPase-activating protein-independent function of the RalBP1/RLIP76 effector

Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.     

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.     

Ral small GTPase signaling and oncogenesis: More than just 15 minutes of fame

Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF–Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.     

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.     

Ral GTPase Down-regulation Stabilizes and Reactivates p53 to Inhibit Malignant Transformation

Ral GTPases are critical effectors of Ras, yet the molecular mechanism by which they induce malignant transformation is not well understood. In this study, we found the expression of K-Ras, RalB, and sometimes RalA, but not AKT1/2 and c-Raf, to be required for maintaining low levels of p53 in human cancer cells that harbor mutant K-Ras and wild-type p53. Down-regulation of K-Ras, RalB, and sometimes RalA increases p53 protein levels and results in a p53-dependent up-regulation of the expression of p21^WAF. K-Ras, RalA, and RalB depletion increases p53 stability as demonstrated by ataxia telangiectasia-mutated kinase activation, increased Ser-15 phosphorylation, and a significant (up to 6-fold) increase in p53 half-life. Furthermore, depletion of K-Ras and RalB inhibits anchorage-independent growth and invasion and interferes with cell cycle progression in a p53-dependent manner. Depletion of RalA inhibits invasion in a p53-dependent manner. Thus, expression of K-Ras and RalB and possibly RalA proteins is critical for maintaining low levels of p53, and down-regulation of these GTPases reactivates p53 by significantly enhancing its stability, and this contributes to suppression of malignant transformation.     

RalB mobilizes the exocyst to drive cell migration

The Ras family GTPases RalA and RalB have been defined as central components of the regulatory machinery supporting tumor initiation and progression. Although it is known that Ral proteins mediate oncogenic Ras signaling and physically and functionally interact with vesicle trafficking machinery, their mechanistic contribution to oncogenic transformation is unknown. Here, we have directly evaluated the relative contribution of Ral proteins and Ral effector pathways to cell motility and directional migration. Through loss-of-function analysis, we find that RalA is not limiting for cell migration in normal mammalian epithelial cells. In contrast, RalB and the Sec6/8 complex or exocyst, an immediate downstream Ral effector complex, are required for vectorial cell motility. RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells. We propose that RalB regulation of exocyst function is required for the coordinated delivery of secretory vesicles to the sites of dynamic plasma membrane expansion that specify directional movement.     

Phosphorylation by protein kinase Cα regulates RalB small GTPase protein activation, subcellular localization, and effector utilization

Ras-like (Ral) small GTPases are regulated downstream of Ras and the noncanonical Ral guanine nucleotide exchange factor (RalGEF) effector pathway. Despite RalA and RalB sharing 82% sequence identity and utilization of shared effector proteins, their roles in normal and neoplastic cell growth have been shown to be highly distinct. Here, we determined that RalB function is regulated by protein kinase Cα (PKCα) phosphorylation. We found that RalB phosphorylation on Ser-198 in the C-terminal membrane targeting sequence resulted in enhanced RalB endomembrane accumulation and decreased RalB association with its effector, the exocyst component Sec5. Additionally, RalB phosphorylation regulated vesicular trafficking and membrane fusion by regulating v- and t-SNARE interactions. RalB phosphorylation regulated vesicular traffic of α5-integrin to the cell surface and cell attachment to fibronectin. In summary, our data suggest that phosphorylation by PKCα is critical for RalB-mediated vesicle trafficking and exocytosis.     

Distinct roles of RalA and RalB in the progression of cytokinesis are supported by distinct RalGEFs

The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras-family protein-dependent and protein-independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.     

Ral GTPases: corrupting the exocyst in cancer 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.     

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

Background

Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.

Methods

Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.

Results

We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.

Conclusions

Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2

In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.     

Ral GTPases in tumorigenesis: Emerging from the shadows

Oncogenic Ras proteins rely on a series of key effector pathways to drive the physiological changes that lead to tumorigenic growth. Of these effector pathways, the RalGEF pathway, which activates the two Ras-related GTPases RalA and RalB, remains the most poorly understood. This review will focus on key developments in our understanding of Ral biology, and will speculate on how aberrant activation of the multiple diverse Ral effector proteins might collectively contribute to oncogenic transformation and other aspects of tumor progression.     

RalA and RalB Proteins Are Ubiquitinated GTPases, and Ubiquitinated RalA Increases Lipid Raft Exposure at the Plasma Membrane

Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.     

Ral家族与肿瘤

Ras相关蛋白Ral家族包括RalA和RalB,参与介导了多种细胞内外信号转导、跨膜运输、细胞形态的维持、转录因子的调节、细胞分化和肿瘤发生与转移等重要的生命活动。Ral家族上游信号通路以及广泛分布的效应器网络对其各种功能起着重要的调控作用,对Ral家族的深入研究将为预防和治疗肿瘤提供了一条新的途径。     

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC₅₀ of 2.2 μM and 2.3 μM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca²⁺ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 μM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.     

Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.     

Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets

Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.