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.
RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.
The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G-protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather than RalA inactivation. Sec5 undergoes phosphorylation in the G-protein binding domain, allosterically reducing RalA interaction. The phosphorylation event is catalyzed by protein kinase C and is reversed by an exocyst-associated phosphatase. Introduction of Sec5 bearing mutations of the phosphorylation site to either alanine or aspartate disrupts insulin-stimulated Glut4 exocytosis, as well as other trafficking processes in polarized epithelial cells and during development of zebrafish embryos. The exocyst thus serves as a “gatekeeper” for exocytic vesicles through a circuit of engagement, disengagement, and re-engagement with G proteins.
The small GTPases RalA and RalB are activated downstream of oncogenic Ras. While activation of RalA is critically important for tumor initiation and growth of Ras-driven cancers, the highly similar small GTPase RalB is implicated in cell survival and metastasis. This difference in function between these two related proteins maps to the C-terminus, a 30 amino acid region that regulates subcellular localization and contains several potential phosphorylation sites. Here we discuss our recent evidence that phosphorylation by the mitotic kinase Aurora A promotes RalA relocalization to mitochondrial membranes, where it recruits the effector RalBP1 and the large dynamin-related GTPase Drp1 to promote mitochondrial fission. As upregulation of both RalA and Aurora A have been observed in human tumors, and phosphorylation of RalA at the site targeted by Aurora A promotes tumorigenesis, it is possible that regulation of mitochondrial fission is one mechanism by which RalA promotes cancer.
Aurora A; cell cycle; mitochondrial fission; RalA; RalBP1
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.
Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases, RalA and RalB, are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole body irradiation but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR.
Methods and Materials
RalA, RalB and their major effectors RalBP1 and Sec5 were knocked down by stable expression of shRNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, γH2AX expression for double-strand DNA breaks (DSBs) and PARP cleavage for apoptosis.
Knockdown of K-Ras, RalA or RalB reduced colony-forming ability post-IR and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR.
Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.
RalA; RalB; RalBP1; pancreatic cancer; ionizing radiation
It is shown that RalA is regulated by a Ral GAP complex (RGC 1/2) in insulin action and links PI 3-kinase signaling to RalA activation. Akt phosphorylates the complex and inhibits its function, resulting in increased RalA activity and glucose uptake.
Insulin stimulates glucose transport in muscle and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.
RLIP76 (RalBP1) is a multidomain protein that is a downstream effector of the small GTP ases RalA and RalB. As well as the Ral binding domain it contains a RhoGAP domain active against Cdc42 and Rac1. RLIP76 also binds to proteins involved in endocytosis and to R-Ras. We recently solved the structure of the Ral binding domain of RLIP76 and the structure of the complex that it forms with RalB. The structure shows that, unlike the other Ral effectors characterized so far, RLIP76 forms a coiled-coil that interacts with RalB. The RLIP76 Ral binding domain binds to both the switch regions of RalB, which are the parts of the G protein that chance conformation upon nucleotide exchange. Here, we review our structure and discuss how it sheds light on the other functions of RLIP76.
G protein; GTPase; RLIP76; RalB; NMR; protein structure
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.
The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.
RalA expression in human prostate cancer is associated with cell migration and is necessary for bone metastasis. However, the downstream effectors of RalA that mediate these functions remain unclear. Here we examined cell migration after small interfering RNA-mediated depletion of Ral effectors Ral binding protein 1 (RalBP1/RLIP), exocyst complex component 2 (Sec5), and phospholipase D1 (PLD1) and found that RalBP1 and RalA depletion inhibited cell migration to a similar extent. Stable lentivirus short hairpin interfering RNA-mediated depletion of RalA and RalBP1 in PC3 human prostate cancer cells inhibited bone metastasis after intracardiac inoculation. Depletion of RalBP1 diminished orthotopic tumor growth of PC3 cells and inhibited spontaneous metastasis from this site. Interestingly, the expression of wild-type or RalA mutants deficient in RalBP1 binding was effective at rescuing the reduced metastatic capacity of RalA-depleted PC3 cells, suggesting that RalA depletion does not reduce this solely by diminished interaction with RalBP1. To determine whether the role of RalBP1 in metastasis is relevant beyond prostate cancer, we studied the requirement of RalBP1 expression in an experimental metastasis model of human bladder cancer, a tumor type with high RalBP1 expression. Depletion of RalBP1 in UMUC3 cells resulted in decreased lung colonization while having a minimal effect on subcutaneous tumor growth. Our studies are the first to suggest that the expression of RalBP1 is necessary for human cancer cell metastasis. Furthermore, we show that the requirement for RalA expression for manifestation of this phenotype is not entirely dependent on a RalA-RalBP1 interaction.
The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G-protein, RalB, is localized to nascent autophagosomes and is activated upon nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.
Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C–dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.
Ras proteins activate Raf and PI-3 kinases, as well as exchange factors for RalA and RalB GTPases. Many previous studies have reported that the Ral signaling cascade contributes positively to Ras-mediated oncogenesis. Here, utilizing a bioengineered tissue model of early steps in Ras-induced human squamous cell carcinoma of the skin, we found the opposite. Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required a knockdown of RalA to a similar degree by shRNA expression in these cells decrease in RalA expression. Moreover, direct ∼2-3 fold knockdown of RalA by shRNA expression in these cells reduced E-cadherin levels and also induced progression to a malignant phenotype. Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues. These phenomena can be explained by our finding that the stability of E-cadherin in Ras-expressing keratinocytes depends upon this RalA signaling cascade. These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.
Up to one third of human melanomas are characterized by an oncogenic mutation in the gene encoding the small GTPase NRAS. Ras proteins activate three primary classes of effectors: Rafs, PI3Ks, and RalGEFs. In melanomas lacking NRAS mutations, the first two effectors can still be activated vis-à-vis oncogenic BRAF mutations coupled with a loss of the PI3K negative regulator PTEN. This suggests that Ras effectors promote melanoma, regardless of whether they are activated by oncogenic NRas. The only major Ras effector pathway not explored for its role in melanoma is the RalGEF-Ral pathway, in which the Ras activation of RalGEFs converts the small GTPases RalA and RalB to an active GTP-bound state. We report that RalA is activated in a number of human melanoma cancer cell lines harboring an oncogenic NRAS allele, an oncogenic BRAF allele, or wild type NRAS and BRAF alleles. Furthermore, shRNA-mediated knock down of RalA, and to a lesser extent RalB, inhibited the tumorigenic growth of melanoma cell lines having these three genotypes. Thus, as is the case for Raf and PI3K signaling, Rals also contribute to melanoma tumorigenesis.
melanoma; RalA; RalB; NRas
The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncogenic proteins in one-third of all human cancers. Oncogenic Ras binds to and activates a number of proteins that promote tumorigenic phenotypes, including the family of Ral guanine nucleotide exchange factors, or RalGEFs. Activated RalGEFs convert the Ral family of small GTPases, comprised of RalA and RalB, from an inactive GDP-bound state to an active GTP-bound state. As both RalA and RalB have been implicated in a variety of tumorigenic phenotypes, we sought to determine which proteins downstream of Rals promote transformation and tumorigenesis. Here we report that shRNA-mediated knockdown of the Ral effector proteins Sec5 and Exo84, but less so in the case of RalBP1, reduced oncogenic RalGEF-mediated transformation and oncogenic Ras-driven tumorigenic growth of human cells. These results suggest that Rals promote oncogenic Ras-mediated tumorigenesis through, at least in part, Sec5 and Exo84.
RalA; RalGEF; Ras; transformation; tumorigenesis
Mutationally activated K-Ras can utilize a multitude of downstream effector proteins to promote oncogenesis. While the Raf and phosphoinositol 3-kinase effector pathways are the best-studied and validated, recent studies have established the critical importance of Ral guanine nucleotide exchange factor (RalGEF) activation of the RalA and RalB small GTPases in cancer biology. Due to recent evidence that the RalGEF-Ral pathway is necessary for the tumorigenic and metastatic potential of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) tumor cells, we investigated whether or not Ral signaling was necessary for KRAS mutant colorectal cancer (CRC) tumor cell growth. As in PDAC, we found upregulated RalA and RalB activation in CRC tumor cell lines and tumors. Surprisingly we found antagonistic roles for RalA and RalB in the regulation of CRC tumor cell anchorage-independent growth. This observation contrasts with PDAC, where RalA but not RalB is necessary for PDAC tumor cell anchorage-independent growth. Our results emphasize cancer cell type differences in Ral function and hence the need for distinct Ral targeted therapeutic approaches in the treatment of CRC vs. PDAC.
Ral guanine nucleotide exchange factors; RalA; RalB; RalBP1; Ras; colorectal cancer; exocyst
3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF. A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.
Metastasis is a complex process during which several gross cellular changes occur. Cells must dissociate from the tumor mass and gain the ability to degrade extracellular matrix and migrate in order to ultimately attach and form a satellite tumor. Regulation of the actin cytoskeleton is an indispensible aspect of cell migration, and many different factors have been implicated in this process. We identified interactions between RalA and its effectors in the Exocyst complex as directly necessary for migration and invasion of prostate cancer tumor cells. Blocking RalA-Exocyst binding caused significant morphological changes and defects in single and coordinated cell migration.
Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.
RalA and RalB are small GTPases which 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. In order 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 demonstrate the clinical relevance of Ral in human cancer and provide a rationale for the development of Ral-directed therapies.
bladder cancer; prostate cancer; squamous cell carcinoma; Ral GTPase; gene expression profiling
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.
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.
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.
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.
metastasis; Ral proteins; invasion; Ral effector mutants; tumor growth
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.
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.
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.
R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.
RalBP1, a multifunctional protein implicated in cancer cell proliferation, radiation and chemoresistance and ligand dependent receptor internalization, is upregulated in bladder cancer and is a downstream effector of RalB, a GTPase associated with metastasis. RalBP1 can be regulated by phosphorylation by protein kinase C (PKC). No studies have comprehensively mapped RalBP1 phosphorylation sites or whether RalB affects these. We identified fourteen phosphorylation sites of RalBP1 in human bladder carcinoma UMUC-3 and embryonic kidney derived 293T cells. The phosphorylated residues are concentrated at the N-terminus. Ten of the first 100 amino acids of the primary structure were phosphorylated. Nine were serine residues, and one a threonine. We evaluated the effect of RalB overexpression on RalBP1 phosphorylation and found the largest change in phosphorylation status at S463 and S645. Further characterization of these sites will provide novel insights on RalBP1 biology, its functional relationship to RalB and possible avenues for therapeutic intervention.
RalBP1; phosphorylation; Ral GTPase