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.     

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.     

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.     

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.     

Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.

Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.     

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.     

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.     

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.     

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 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.     

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 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.     

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.     

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.     

ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA

Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa. Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell morphology, and effects on adherence processes. Preferred substrates of the ADP-ribosyltransferase (ADPRT) portion of ExoS include low molecular weight G-proteins (LMWG-proteins) in the Ras family. In examining the ADP-ribosylation and functional effects of ExoS on RalA, ExoS was found to ADP-ribosylate endogenous RalA and recombinant RalADeltaCAAX at multiple sites, with Arg52 identified as the preferred site of ADP-ribosylation. The binding of RalA to the Ral binding domain (RBD) of its downstream effector, RalBP1, was inhibited by bacterially translocated ExoS, indicating an effect of ExoS on cellular RalA function. In vitro analyses confirmed that ADP-ribosylation of RalA directly interfered with its ability to bind to the RBD of RalBP1. The studies support the fact that RalA is a cellular substrate of bacterially translocated ExoS and that ADP-ribosylation by ExoS affects RalA interaction with its downstream effector, RalBP1.     

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.     

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.     

The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene.

Ral is a small GTPase involved in critical cellular signaling pathways. The two isoforms, RalA and RalB, are widely distributed in different tissues, with RalA being enriched in brain. The best characterized RalA signaling pathways involve RalBP1 and phospholipase D. To investigate RalA signaling in neuronal cells we searched for RalA-binding proteins in brain. We found at least eight proteins that bound RalA in a GTP-dependent manner. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified these as the components of the exocyst complex. The yeast exocyst is a regulator of polarized secretion, docking vesicles to regions of the plasma membrane involved in active exocytosis. We identified the human FLJ10893 protein as the mammalian homologue of the yeast exocyst protein Sec3p. The exocyst complex did not contain the previously identified exocyst component rSec15, but a new homologue of both yeast Sec15p and rSec15, called KIAA0919. Western blots confirmed that two rat exocyst proteins, rSec6 and rSec8, bound active RalA in nerve terminals, as did RalBP1. Phospholipase D bound RalA in a nucleotide-independent manner. This places the RalA signaling system in mammalian nerve terminals, where the exocyst may act as an effector for activated RalA in directing sites of exocytosis.     

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells

RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.     

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.