Small GTPase RalA Research Articles

RalA promotes a direct exocyst-Par6 interaction to regulate polarity in neuronal development.

Cell polarization is essential for neuronal development in both the embryonic and postnatal brain. Here, using primary cultures, in vivo postnatal electroporation and conditional genetic ablation, we show that the Ras-like small GTPase RalA and its effector, the exocyst, regulate the morphology and polarized migration of neural progenitors derived from the subventricular zone, a major neurogenic niche in the postnatal brain. Active RalA promotes the direct binding between the exocyst subunit Exo84 and the PDZ domain of Par6 through a non-canonical PDZ-binding motif. Blocking the Exo84-Par6 interaction impairs polarization in postnatal neural progenitors and cultured embryonic neurons. Our results provide the first in vivo characterization of RalA function in the mammalian brain and highlight a novel molecular mechanism for cell polarization. Given that the exocyst and the Par complex are conserved in many tissues, the functional significance of their interaction and its regulation by RalA are likely to be important in a wide range of polarization events.

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

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

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

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

PGE2 promotes renal carcinoma cell invasion through activated RalA

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

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

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

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

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

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

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

Aurora Mitochondrialis Drives Fission during Mitosis

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

RALA and RALBP1 regulate mitochondrial fission at mitosis

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

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

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

Cyclin D1 interacts and collaborates with Ral GTPases enhancing cell detachment and motility

Alterations in the levels of adhesion and motility of cells are critical events in the development of metastasis. Cyclin D1 (CycD1) is one of the most frequently amplified oncogenes in many types of cancers and it is also associated with the development of metastasis. Despite this, we still do not know which are all the relevant pathways by which CycD1 induces oncogenic processes. CycD1 functions can be either dependent or independent of the cyclin-dependent kinase Cdk4, and they affect several cellular aspects such as proliferation, cell attachment and migration. In this work, we reveal a novel function of CycD1 that fosters our understanding of the oncogenic potential of CycD1. We show that CycD1 binds to the small GTPases Ral A and B, which are involved, through exocyst regulation, in the progression of metastatic cancers, inducing anchorage-independent growth and cell survival of transformed cells. We show that CycD1 binds active Ral complexes and the exocyst protein Sec6, and co-localizes with Ral GTPases in trans-Golgi and exocyst-rich regions. We have also observed that CycD1-Cdk4 phosphorylates the Ral GEF Rgl2 'in vitro' and that CycD1-Cdk4 activity stimulates accumulation of the Ral GTP active forms. In accordance with this, our data suggest that CycD1-Cdk4 enhances cell detachment and motility in collaboration with Ral GTPases. This new function may help explain the contribution of CycD1 to tumor spreading.

A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

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.

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

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. RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expression of short hairpin RNAs 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, gammaH2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (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 the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.

Salmonella-Directed Recruitment of New Membrane to Invasion Foci via the Host Exocyst Complex

Salmonella attachment to the intestinal epithelium triggers delivery of bacterial effector proteins into the host cytosol through a type III secretion system (T3SS), leading to pronounced membrane ruffling and macropinocytic uptake of attached bacteria. The tip of the T3SS is made up of two proteins, SipB and SipC, which insert into the host plasma membrane, forming a translocation pore. Both the N and C termini of SipC are exposed in the host cytosol and have been shown to directly modulate actin cytoskeleton assembly. We have identified a direct interaction between SipC and Exo70, a component of the exocyst complex, which mediates docking and fusion of exocytic vesicles with the plasma membrane. Here, we show that exocyst components coprecipitate with SipC and accumulate at sites of invasion by Salmonella typhimurium. Exocyst assembly requires activation of the small GTPase RalA, which we show is triggered during Salmonella infection by the translocated effector, SopE. Knockdown of RalA or Sec5 results in reduced membrane ruffling at sites of attachment and impairs bacterial entry into host cells. These findings suggest that S. typhimurium enhances invasion efficiency by promoting localized membrane expansion, directly through SipC-dependent recruitment of the exocyst and indirectly via SopE-dependent activation of RalA.

Ral activation promotes melanomagenesis

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.

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

The Small GTPase Ral Couples the Angiotensin II Type 1 Receptor to the Activation of Phospholipase C-δ1

The angiotensin II type 1 receptor (AT(1)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1)R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) beta via its association with G alpha(q/11), leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT(1)R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT(1)R, but that RalA expression is required for AT(1)R-stimulated IP formation but not 5-HT(2A) receptor-mediated IP formation. AT(1)R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the beta-arrestin-dependent plasma membrane translocation of RalGDS. G alpha(q/11) small interfering RNA (siRNA) treatment also significantly attenuates both AT(1)R- and 5-HT(2A) receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-delta1 has been reported to be activated by RalA, and we show that AT(1)R-stimulated IP formation is attenuated after PLC-delta 1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-delta 1 stimulation.

RalA-Exocyst Complex Regulates Integrin-Dependent Membrane Raft Exocytosis and Growth Signaling

Anchorage dependence of cell growth is a key metastasis-suppression mechanism that is mediated by effects of integrins on growth signaling pathways. The small GTPase RalA is activated in metastatic cancers through multiple mechanisms and specifically induces anchorage independence. Loss of integrin-mediated adhesion triggers caveolin-dependent internalization of cholesterol- and sphingolipid-rich lipid raft microdomains to the recycling endosomes; these domains serve as platforms for many signaling pathways, and their clearance from the plasma membrane (PM) after cell detachment suppresses growth signaling. Conversely, readhesion triggers their return to the PM and restores growth signaling. Activation of Arf6 by integrins mediates exit of raft markers from the recycling endosomes but is not sufficient for return to the PM. We now show that RalA but not RalB mediates integrin-dependent membrane raft exocytosis through the exocyst complex. Constitutively active RalA restores membrane raft targeting to promote anchorage-independent growth signaling. Ras-transformed pancreatic cancer cells also show RalA-dependent constitutive PM raft targeting. These results identify RalA as a key determinant of integrin-dependent membrane raft trafficking and regulation of growth signaling. They therefore define a mechanism by which RalA regulates anchorage dependence and provide a new link between integrin signaling and cancer.

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Author SummaryNeurons adapt over time in order to dampen their response to prolonged or particularly strong stimuli. This process, termed long-term depression (LTD), results in a long-lasting decrease in the efficiency of synaptic transmission. One extensively studied form of LTD requires the activation of a specific class of receptors known as NMDA glutamate receptors (NMDARs). A key molecular event initiated by NMDA receptor activation is the stimulation of protein phosphatases. Another key event is internalization via endocytosis of synaptic AMPA glutamate receptors (AMPARs). However, the mechanism by which protein dephosphorylation is coupled to AMPAR endocytosis has remained unclear. Here, we help to define this mechanism. We show that endocytic proteins, including RalBP1, are widely distributed in neurons under normal conditions. Upon NMDAR activation, the small GTPase RalA becomes activated and binds to RalBP1, resulting in the translocation of RalBP1 and RalBP1-associated endocytic proteins to synapses. At the same time, RalBP1 becomes dephosphorylated, which promotes its binding to the postsynaptic scaffold protein PSD-95, a protein that itself associates with AMPARs. This concerted recruitment of endocytic proteins to the vicinity of AMPARs results in AMPAR endocytosis. On the basis of our data, we propose a model in which dual binding of RalBP1 to both RalA and PSD-95 following RalBP1 dephosphorylation is essential for NMDAR-dependent AMPAR endocytosis during LTD.

Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.