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.     

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.     

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.     

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.     

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 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.     

Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth

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.     

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.     

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.     

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-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.     

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.     

Dual effects of Ral-activated pathways on p27 localization and TGF-β signaling

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 p27^Kip1 (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.     

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。     

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.     

Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-δ1

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-δ1, and increases its enzymatic activity. Since PLC-δ1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-δ1. Therefore, we created a GST-PLC-δ1 construct consisting of the first 294 amino acids of PLC-δ1 (GST-PLC-δ1_1-294). In vitro binding experiments confirmed that PLC-δ1_1-294 was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-δ1, demonstrating that PLC-δ1 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-δ1 were able to inhibit RalA binding to PLC-δ1_1-294. This study demonstrates that there are two binding sites for RalA in PLC-δ1 and provides further insight into the role of Ral GTPase in the regulation of PLC-δ1 function.     

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

Background

RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.

Methodology/Principal Findings

We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca²⁺ and cAMP and is prevented by the L-type voltage-gated Ca²⁺ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.

Conclusions/Significance

Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca²⁺ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.     

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.