InteractiveFly: GeneBrief

Ras-related protein: Biological Overview | References


Gene name - Ras-related protein

Synonyms - Ral

Cytological map position - 3E5-3E6

Function - signaling

Keywords - peripheral nervous system, regulation of exocyst protein complex, negative regulator of JNK activity

Symbol - Rala

FlyBase ID: FBgn0015286

Genetic map position - X: 3,599,017..3,613,665 [-]

Classification - Ral (Ras-like) subfamily

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Holly, R. M., Mavor, L. M., Zuo, Z. and Blankenship, J. T. (2015). A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo. Development 142: 2316-2328. PubMed ID: 26092850
Summary:
Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. This study used 4D imaging to identify furrow formation, stabilization, and regression periods, and identified a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. It was demonstrated that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

Tracy, K., Velentzas, P. D. and Baehrecke, E. H. (2015). Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner. EMBO Rep [Epub ahead of print]. PubMed ID: 26598552
Summary:
Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. This study investigated Ral function in different cellular contexts in Drosophila and found that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.

Klose, M., Duvall, L. B., Li, W., Liang, X., Ren, C., Steinbach, J. H. and Taghert, P. H. (2016). Functional PDF signaling in the Drosophila circadian neural circuit is gated by Ral A-dependent modulation. Neuron [Epub ahead of print]. PubMed ID: 27161526
Summary:
The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. This study reports daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn. The rhythm persists in constant darkness and does not require endogenous ligand (PDF) signaling or rhythmic receptor gene transcription. Furthermore, rhythmic responsiveness reflects the properties of the pacemaker cell type, not the receptor. Dopamine responsiveness also cycles, in phase with that of PDF, in the same pacemakers, but does not cycle in large LNv. The activity of RalA GTPase in s-LNv regulates PDF responsiveness and behavioral locomotor rhythms. Additionally, cell-autonomous PDF signaling reversed the circadian behavioral effects of lowered RalA activity. Thus, RalA activity confers high PDF responsiveness, providing a daily gate around the dawn hours to promote functional PDF signaling.
Richhariya, S., Jayakumar, S., Abruzzi, K., Rosbash, M. and Hasan, G. (2017). A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons. Sci Rep 7: 42586. PubMed ID: 28195208
Summary:
Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrate a requirement for SOCE in neurons that regulate Drosophila flight bouts. This study refined this requirement temporally to the early pupal stage and used RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervous system. Down regulation of dStim, the endoplasmic reticular calcium sensor and a principal component of SOCE in the nervous system, alters the expression of 131 genes including Ral, a small GTPase. Disruption of Ral function in neurons impairs flight, whereas ectopic expression of Ral in SOCE-compromised neurons restores flight. Through live imaging of calcium transients from cultured pupal neurons, it was confirmed that Ral does not participate in SOCE, but acts downstream of it. These results identify neuronal SOCE as a mechanism that regulates expression of specific genes during development of the pupal nervous system and emphasizes the relevance of SOCE-regulated gene expression to flight circuit maturation.



BIOLOGICAL OVERVIEW

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).

The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors (Hamad, 2002; Rangarajan, 2004). Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture (Gonzalez-Garcia, 2005). The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).

None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis (Jullien-Flores, 2000; Nakashima, 1999). The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking (Agapova, 2004; Chien, 2003; Essers, 2004; Goi, 2000; Henry, 2000; Kops, 1999) with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).

In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).

Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral (Mirey, 2003; Sawamoto, 1999a). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).

The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).

Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of RalG20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).

The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).

Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999b), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation (de Ruiter, 2000; Essers, 2004). The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).

Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).

A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).

The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).

Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts (Kennedy, 2003). The current results can explain one of the contributions of the Ral pathway to oncogenesis (Gonzalez-Garcia, 2005; Urano, 1996; White, 1996): cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).

Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however, their role in a developing organism is poorly understood. This study identified the Drosophila Ral homologue RalA as a new key regulator of polar-cell differentiation during oogenesis. Polar cells have a crucial role in patterning the egg chamber and in recruiting border cells, which undergo collective and guided migration. RalA function is essential for the maintenance of anterior and posterior polar-cell fate and survival. RalA is required cell autonomously to control the expression of polar-cell-specific markers, including the Jak/Stat ligand Unpaired. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result, border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus, RalA is required in organizing centres to control proper patterning and migration in vivo (Ghiglione, 2008).

RalA shows a cell non-autonomous phenotype originating from the PC. PCs are essential anteriorly for recruiting a ring of around six outer border cells (oBCs) that make a mature BC cluster, which depends on the secretion of the Unpaired (Upd) ligand from the PC and subsequent Jak/Stat activation in the oBC. Secretion of Upd and binding to Domeless (Dome), the Drosophila Jak/Stat receptor, induces ligand-dependent internalization of Dome in cells surrounding PCs, both in BCs and posterior follicle cells. When PCs were mutant for RalA, Dome-containing endocytic vesicles were no longer observed, both anteriorly and posteriorly, suggesting that Jak/Stat signalling was not activated. In wild-type egg chambers, Stat is localized in the nucleus as a gradient, with higher levels of nuclear Stat close to the PC, thus reflecting Jak/Stat pathway activation. The nuclear localization of Stat was normal when PCs were wild type with adjacent follicle cells mutant for RalA, indicating that RalA does not have a role in the function of oBCs and posterior follicle cells in controlling Jak/Stat signalling. By contrast, in egg chambers with mutant PCs, Stat nuclear localization was completely abolished. To discriminate between a role of RalA in Upd expression or activity, egg chambers were stained using a Upd antibody, which shows a gradient of this ligand in egg chambers. Using this assay, it was shown that RalA mutations in PCs strongly affect the expression of the Upd protein (Ghiglione, 2008).

Does the reduction of Upd lead to non-autonomous defects posteriorly? It was shown previously that the Jak/Stat pathway is essential for specifying posterior follicle cells, which then signal back to the oocyte for anterior–posterior polarization. When polarity is normal, the Staufen protein forms a posterior crescent in the oocyte. In egg chambers containing posterior RalA mutant PCs, the localization of Staufen was not normal and it was found centrally in strongly affected oocytes, similar to mutants that fail to reorganize the microtubules (Ghiglione, 2008).

Among follicle cells, PCs have been shown to be important in patterning the egg chamber and in establishing BCs. This study identified RalA as a new key regulator of PC fate. RalA is essential both cell autonomously for maintaining PC differentiation and cell non-autonomously for patterning terminal follicle cells through Jak/Stat signalling. However, RalA mutations do not reproduce the full range of Jak/Stat mutations, consistent with the fact that some Upd is still produced by RalA mutant PCs. For example, the follicle cell markers MA33 and dpp-lacZ, the expression of which in stretched cells is controlled by Jak/Stat signalling, are expressed normally when PCs are mutant for RalA. Altogether, these data indicate that the function of RalA is essential for maintaining the PC fate and for ensuring high levels of Upd expression, which are required for patterning the most terminal follicle cells, including BCs and posterior follicle cells. The RalA phenotype suggests the existence of a maintenance signal taking place around stage 6-7 -- that is, following egg chamber proliferation phase -- which would be necessary to complete egg chamber patterning by providing sustained Jak/Stat activation (Ghiglione, 2008).

Previous studies have shown that Ral proteins interact with Sec5 and Exo84 to regulate the exocyst function during proliferation and tumorigenesis. The current in vivo study suggests a role for RalA in cell differentiation and patterning, independent of secretion. Not only Upd but also several non-secreted PC markers lose expression following RalA loss of function, reminiscent of a more general differentiation phenotype. Contrary to what would be expected of a secretion phenotype, the Upd protein does not accumulate within PCs that are mutant for RalA. The analysis of sec5 mutations in the follicle cells showed that this gene is required for the positioning of the oocyte and for follicle cell morphology, two phenotypes that were never observed in RalA mutant egg chambers. Finally, expression in BCs of RNA-mediated interference against the sec5, sec6, sec8 or sec15 genes did not show any phenotype (Ghiglione, 2008).

Thus, instead of showing a ubiquitous activity, the data indicate a cell-type-specific function for RalA in PCs, independent of secretion. Controlling the differentiation of PCs, which have a central organizing role through Jak/Stat ligand production, might represent a way to monitor the number of invasive cells during both normal development and tumour cell invasion. Interestingly, in mouse M1 myeloid leukaemia cells, Stat3 can activate Ral by controlling the expression of its exchange factor. These data suggest a conserved functional link between Ral proteins and Stat activity and provide a basis for the maintenance of Jak/Stat activity in PCs through a positive feedback loop involving RalA and Stat (Ghiglione, 2008).

A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways

Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. This study revisited the Ras/Rap-Ral connections in Drosophila by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. It was shown that (1) the Ral-centered protein network appears to be extremely conserved in human and flies, (2) in this network, Ral guanine nucleotide exchange factor 2 (RGL) is a functional Drosophila orthologue of RalGEFs, and (3) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. These data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities (Mirey, 2003).

The functions of Ral proteins remain unclear. They are not oncogenic per se, but they facilitate Ras transformation, participate in cell motility, and are required for metastatic evolution of Ras-transformed cells as well as for Ras-induced stimulation of cyclin D1 expression. They are involved in phospholipase D activation, endocytosis, and exocytosis. There is not yet a unifying theory that relates these latter functions to the former cancer-connected phenotypes (Mirey, 2003 and references therein).

In mammalian cell lines, Ral proteins were shown to be involved in not only H-Ras and K-Ras but also TC21 signaling via a family of Ras effectors, the RalGEFs. Once Ras is bound to GTP, it binds and activates these RalGEFs, which in turn activate Ral proteins. There are also Ras-independent pathways that activate Ral (Mirey, 2003 and references therein).

Rap proteins are GTPases once described as antagonistic to Ras oncoproteins. Their function remains elusive. They were reported to be functionally connected to integrin signaling, and they are able to bind RalGDS, one of the mammalian RalGEFs, with a higher affinity than Ras, yet this interaction does not lead to the activation of Ral in cell lines (Mirey, 2003 and references therein).

This study addressed the following questions: (1) The contribution of the Ral pathway to cellular functioning was examined by using approaches with different methodological biases; (2) Within the frame of a whole organism it was asked where cells have to communicate with neighboring cells of different types and integrate various signals; (3) It was desirable to have several readouts, assuming that signaling pathways might be using signaling modules following different architectures in different situations; and (4) The power of genetics was used to establish signaling cascades as well as functional interactions between distinct signaling pathways (Mirey, 2003).

First, it was shown that an exchange factor for Ral of the RalGEF family, which is an orthologue of mammalian RGL, exists in Drosophila. In fact, flies express two orthologues, RGL1 and RGL2, probably generated by the use of two promoters and alternative splicing. RGL1 and RGL2 share the same RalGEF domain as well as the C-terminal domain that binds Ras and Rap, but they differ in their N termini. Combined data from several two-hybrid screens, including the present one, suggest that the Ras/Rap-Ral network is very similar in mammals and in Drosophila. Physical interactions connect RGL to RAS1 (Ras in humans), RAS2 (R-Ras and/or TC21 in humans), and RAS3 (Rap1 in humans) as well as RAL to RLIP (RLIP76 in humans) and SEC5 (the same in humans). RLIP is connected to the orthologous µ2 chains of the AP2 complexes as well as to REPS (the same in humans). The conservation of such a large network confirms that Drosophila is a suitable model to study the Ral pathway in a physiological context. It is noteworthy that in Caenorhabditis elegans, all the proteins of this network exist and certain interactions have been shown, as opposed to what is seen in S. cerevisiae, suggesting that they are important for metazoans (Mirey, 2003).

Several lines of transgenic flies were generated to decipher the functional relationships between the different actors. Phenotypes of the transgenic flies suggest that, like in mammals, the function of Rgl is not totally accounted for by the activation of Ral, since an activated allele of Ral does not mimic the activated alleles of Rgl. Could activated Rgl phenotypes be due to the titration of endogenous RAS1 or RAP1 by the RA domain of the RGL transgenes? If so, coexpression of activated RGL with either wild-type Ras1 or wild-type Rap1 should attenuate the Rgl phenotypes. This is not the case. Flies coexpressing activated RGL and RAS1 display some new phenotypes which are not seen when each transgene is individually expressed (extra veins under en-GAL4; heterogeneity of ommatidia size under GMR-GAL4) or keep displaying the Rgl phenotype (bristle morphology under sca-GAL4). Flies coexpressing activated RGL and RAP1 even display an enhanced Rgl phenotype (in eyes and on wings) or keep displaying the bristle morphology phenotype due to activated RGL. Ral-independent functions of RGL might be mediated by protein-protein interactions with domains other than the Ras/Rap and Ral interacting domains, and recently, mammalian RalGDS was shown to interact with ß-arrestin. However the titration hypothesis cannot be ruled out totally. An alternative explanation might be that Ral has to cycle between a GDP state and a GTP state, which would be accelerated by activated RalGEF and not mimicked by activated Ral that is blocked in a GTP-bound state. Although the existence of Ral-independent functions of RalGEFs is suggested both in mammals and in flies, the clarification of this question requires further investigation (Mirey, 2003).

But is RGL an actual exchange factor for RAL? The effects in bristle development of a dominant-negative Ral are suppressed by the increased expression of Rgl. The simplest explanation is that RGL is a bona fide exchange factor for Ral (Mirey, 2003).

Interactions were investigated between the Ral pathway and two of its interlocutors, the Ras and Rap GTPases. In mammalian cell lines, the RasG12V E37G, RasG12V Y40C, and RasG12V T35S alleles activate the Ral pathway via interaction with RalGEFs, the PI3K pathway, and the Raf pathway, respectively, although things might be more complicated since RasG12V Y40C might be acting together with RasG12V E37G to activate RalGEFs upon epidermal growth factor stimulation (Tian, 2002). RasG12V E37G does not activate the Raf nor the PI3K pathway. In Drosophila also, these different Ras alleles drive different pathways; however, nothing is known about the connection between the Ras and Ral pathways (Mirey, 2003).

If a Ras-Ral pathway exists, a dominant-negative allele of Ral should attenuate effects due to RasG12V E37G but not phenotypes due to RasG12V Y40C or RasG12V T35S. Indeed, in HeLa cells, dominant-negative alleles of Ral do block a RasG12V E37G phenotype (Ikeda, 1998). Reciprocally, RasG12V E37G, but not the two other alleles, might attenuate a Ral dominant-negative phenotype. The two-hybrid results show that, as in mammals, fly RGL behaves as an effector of fly RAS1, and this interaction is mediated by the RA domain of RGL. When searching for genetic interactions between Ras1 and Ral, it was found that all three Ras alleles enhanced the RalS25N loss-of-bristle phenotype. These results show an actual genetic interaction between the Ras and Ral pathways but do not support the classical model of a linear pathway from Ras to Ral, a conclusion strengthened by the absence of the Ras allele specificity of the observed interactions. An alternative model would be an intersection of the Ral and Ras pathways and would involve a yet undefined Ras effector whose interaction with Ras would not be selective for the three effector loop mutations tested in this study (Mirey, 2003).

Rap1 is another GTPase of the Ras family that can interact with most Ras effectors, including RalGEFs. No functional Rap1-RalGEF, Rap1-PI3K, or Rap1-Raf interactions have been documented, except for an isoform of B-Raf, described as activated by Rap1. The originally suggested antagonism between Ras and Rap remains a murky issue, and Rap1 and Ras seem to participate in rather independent pathways, although recent data challenge this idea, at least in vesicle trafficking at synapses. In Drosophila, where Rap1 is required for morphogenesis, Ras1 and Rap1 act in distinct pathways (Asha, 1999). No functional effector of Rap has been identified. Two-hybrid data show that Drosophila RGL behaves as a Rap1 effector. Genetic data support the idea that this interaction is functional: a dominant-negative allele of Ral is able to rescue lethality caused by an activated allele of Rap1, and reciprocally, in the surviving flies, activated Rap1 rescues the bristle development phenotype of a dominant-negative Ral. Similarly, in eyes, Rgl and Ral seem to act downstream of Rap1. Although an alternative model where Rap and Ral signals converge towards a common downstream target cannot be ruled out, the results rather argue in favor of a linear Rap-Rgl-Ral pathway. Consistent with this model, preliminary data with an engrailed-GAL4 driver show that the phenotype displayed by RalS25N in wings mimics the one obtained by overexpression of a negative regulator of Rap, RapGAP. Thus, titrating RGL proteins by the expression of a dominant-negative Ral mimics the inactivation of Rap1 by an excess of its GAP (Mirey, 2003).

Taken together, the genetic data from Drosophila shed a different light on signaling networks as they were established in mammalian cell lines. In both developmental systems used in this study (eye and notum), Ras1 and Ral do not seem to be linearly connected. In contrast, Rap1 and Ral are shown to act as if they were participating in a common transduction pathway. These data do not rule out that in some other tissues, a Ras-Ral pathway might indeed exist, but they suggest that a molecular Lego might assemble signaling modules following various architectures in different tissues. It is speculated that this should also be the case in mammals. An alternative model would be that a Ras-Ral pathway cannot be revealed in this experimental system, just as the Rap-Ral pathway couldn't be revealed in mammalian cell lines, and that, in the same tissue, Ras-Ral and Rap-Ral pathways are functional (Mirey, 2003).

Identification and expression of Ima, a novel Ral-interacting Drosophila protein

This study reports the identification of Ima (FlyBase name: Magi), a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays. The gene is expressed in distinct spatiotemporal patterns throughout embryonic development. Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner (Beller, 2002).

The finding that ima is expressed in the posterior terminal region of the embryo, which is established in response to torso-dependent Ras/Raf signaling, prompted an inquiry as to whether and how ima expression is controlled by the activity of this pathway. ima expression was examined in embryos that lack the activity of key components of the maternal terminal organizer system or its mediators such as the terminal gap genes tailless and huckebein. Embryos from females which were homozygous for the torso loss-of-function allele torpm failed to express the posterior terminal ima expression domain, whereas the dorsal expression domain of ima was not affected. This result shows that activated components of the torso signaling pathway are necessary for the activation of posterior terminal ima gene expression (Beller, 2002).

In order to place ima within the torso-dependent signaling cascade, it was asked whether ima is a direct control target of the maternal components of the pathway or whether its expression is regulated in response to their zygotic mediators, namely the zygotic gap genes tailless and huckebein. Posterior terminal expression was unchanged in embryos homozygous for the tailless loss-of-function allele taillessl10–22 but absent from embryos homozygous for a huckebein loss-of-function allele (hkb2). Posterior terminal ima expression is not affected in homozygous hindsight mutant embryos, which lack a transcription factor that acts downstream and in response to huckebein activity. torso-dependent control of ima expression is therefore mediated by huckebein activity and represents a direct or indirect target of the huckebein encoded transcription factor and does not require hindsight activity (Beller, 2002).

In order to test whether lack of ima activity interferes, for example, with huckebein-dependent aspects of embryonic development, homozygous embryos were examined for the deletion Df(2R)CC2 which uncovers the genomic region 2R 57C2-57C5 as assessed by in situ hybridization to polytene salivary gland chromosomes. It deletes ima (2R 57C2,3) as well as several other genes. Homozygous Df(2R)CC2 embryos develop no obvious body patterning defects but die in the egg shell. These observations indicate that the lack of ima does not cause a scorable morphological defect (Beller, 2002).

ima gain-of-function experiments were performed by employing the Gal4/UAS system to examine the effect of a UAS-dependent Ima expression. Using the T80-Gal4 and daG32-Gal4 drivers, ima was ubiquitously expressed. In addition, ima was expressed in en-Gal4-driven stripes along the longitudinal axis of gastrulating embryos and by responding to the C155 elav-Gal4 driver in neurons. en stripe expression of ima had no scorable effect on development, whereas ubiquitous and neural overexpression of Ima consistently caused a rough eye phenotype and supernumerary, misoriented bristles on the notum. None of these effects were observed when Ima deletion mutants lacking either the WW domains or the PDZ domains were expressed under otherwise identical conditions as full-size Ima. Collectively, these observations indicate that misexpression of Ima interferes in a tissue-specific manner with normal Drosophila development and that this activity requires both the WW and the PDZ domains (Beller, 2002).

Novel guanine nucleotide exchange factor GEFmeso of Drosophila melanogaster interacts with Ral and Rho GTPase Cdc42

A DBL-like guanine nucleotide exchange factor (GEF) in Drosophila, called GEFmeso (CG30115), has been identified a novel binding target of the Ras-like GTPase Ral. Previous studies suggested that some aspects of Ral activity, which is involved in multiple cellular processes, are mediated through regulation of Rho GTPases. This study shows in vitro association of GEFmeso with the GTP-bound active form of Ral and the nucleotide-free form of the Rho GTPase Cdc42. GEFmeso fails to bind to other Rho GTPases, showing that Cdc42 is a specific interaction partner of this GEF. Unlike Ral and Cdc42, which are ubiquitously expressed, GEFmeso exerts distinct spatio-temporal expression patterns during embryonic development, suggesting a tissue-restricted function of the GEF in vivo. Based on previous observations that mutations in Cdc42 or overexpression of mutant alleles of Cdc42 lead to distinct effects on wing development, the effects of overexpression of dominant-negative and activated versions of Ral on wing development were analyzed. In addition, GEFmeso overexpression studies as well as RNAi experiments were performed. The results suggest that Ral, GEFmeso and Cdc42 act in the same developmental pathway and that GEFmeso mediates activation of Cdc42 in response to activated Ral in the context of Drosophila wing development (Blanke, 2006).

GEFmeso encodes at least two transcripts of different sizes. The longer transcript contains a N-terminal DH and PH domain in an arrangement that is characteristic for GEFs of the DBL family. GEFmeso is conserved in other insects such as Anopheles, but no direct vertebrate homologue could be identified. It interacts with constitutively active DRalG20V protein and, to a lower degree, with the dominant-negative mutant DRalS25N. Deletion analysis indicates that the Pro707-Ser830 interval of GEFmeso is the core region, which mediates DRal binding. This sequence interval lacks similarity to the Ral binding regions of previously identified mammalian Ral binding proteins. It is noteworthy that these other proteins also fail to have a common sequence motif for Ral binding (Blanke, 2006).

Ral has been implicated in the regulation of the cytoskeleton reorganization required for cell migration and cell shape changes (Ohta, 1999; Gildea, 2002; Suzuki, 2000). Both processes are also dependent on Rho GTPase activities. Furthermore, the Ral effector protein RLIP76/RalBP1/RIP1 (Jullien-Flores, 1995; Cantor, 1995; Park, 1995) functions as a GAP for Cdc42 and Rac, respectively. These earlier results imply that the biological response to Ral activity could be mediated by these Rho GTPase activities. The current results provide additional evidence for a molecular link between DRal and Rho GTPase activity. GEFmeso binds to activated DRal and specifically associates with only one of the Rho-like GTPases of Drosophila, i.e., the nucleotide-depleted DCdc42. The finding that both DRal and DCdc42 are in vitro binding targets of GEFmeso and the fact that loss-of-function and gain-of-function experiments with DRal and GEFmeso transgenes in the wing result in DCdc42-like wing phenotypes suggest that both DRal and GEFmeso may act either in the same or a parallel genetic circuitry as DCdc42 (Blanke, 2006).

The expression of GEFmeso is restricted to distinct regions of the developing embryo including the prospective mesodermal region on the ventral side of the blastoderm embryo and a stripe pattern in the developing mesoderm as well as growing imaginal discs of the larvae. In contrast DRal and DCdc42 are ubiquitously expressed, suggesting that DRal and DCdc42 functions are spatio-temporally regulated by restricted expression of mediators like GEFmeso. In this context it is noteworthy to mention that the number of both GEFs and possible Rho GTPase substrates are increased during evolution, and that individual GEFs become more specialized in higher eukaryotes. For example, many of the mammalian DBL family members exert tissue- and cell type-specific expression patterns and can act on distinct subsets of Rho GTPases or on a single specific target Rho GTPase. This evolutionary mechanism allows further diversification of even those signaling pathways that are based on ubiquitously expressed key signaling molecules like, for example, small GTPases, which are in this way involved in diverse cellular processes (Blanke, 2006).

During gastrulation GEFmeso is likely to be required for the mesoderm invagination process. This conclusion is based on the finding that ubiquitous GEFmeso activity prior to and during gastrulation of the embryo impairs this process severely. Although it is not known whether this effect involves DRal and/or DCdc42 activities, the result underscores the need for a strictly regulated expression of GEFmeso and argues for a function of GEFmeso as a signaling component exerting spatio-temporal restricted activation of target proteins (Blanke, 2006).

The combined results are consistent with the proposal that GEFmeso acts as a spatio-temporal restricted signaling component that mediates DRal activity and provides a direct link between activated DRal and a downstream DCdc42-dependent developmental process. It is not known yet whether the DRal-GEFmeso-DCdc42 pathway is also linked to the previously established Ras-RalGDS-Ral ( Wolthuis, 1999) or Rap-RGL-Ral (Mirey, 2003) signaling pathways. Nevertheless, the DRal-GEFmeso-DCdc42 cascade provides another example of a signaling pathway, where multiple small GTPases are linked by GEFs within one signaling cascade (Blanke, 2006).

Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. A Drosophila homolog of Ral has been identified that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal does not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants cause defects in the development of various tissues, including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein causes defects in the development of hairs and bristles. These phenotypes are genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH(2)-terminal kinase kinase (JNKK) and Jun NH(2)-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein causes defects in the process of dorsal closure during embryogenesis and inhibits the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway (Sawamoto, 1999a; full text of article).

The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH(2)-terminal kinase pathway

The small GTP-binding protein Ral is activated by RalGDS, one of the effector molecules for Ras. Active Ral binds to a GTPase activating protein for CDC42 and Rac. Although previous studies have suggested a role for Ral in the regulation of CDC42 and Rac (two proteins involved in arranging the cytoskeleton), Ral's in vivo function is largely unknown. To examine the effect on development of overexpressing Ral, transgenic Drosophila were generated that overexpress wild-type or mutated Ral during eye development. While wild-type Ral causes no developmental defects, expression of a constitutively activated protein results in a rough eye phenotype. Activated Ral does not affect cell fate determination in the larval eye discs but causes severe disruption of the ommatidial organization later in pupal development. Phalloidin staining shows that activated Ral perturbs the cytoskeletal structure and cell shape changes during pupal development. This phenotype is similar to that caused by RhoA overexpression. In addition, the phenotype is synergistically enhanced by the coexpression of RhoA. These results suggest that Ral functions to control the cytoskeletal structure required for cell shape changes during Drosophila development (Sawamoto, 1999b; full text of article).

Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation

Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. This study shows that in response to planar cell polarity (PCP) signals, Ral modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. This study discovered a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, it was shown that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation (Cho, 2011).

These results presented support a model for Ral function (see Model for Ral function in R3/R4 cell fate decision). Ral transcription is upregulated in response to Fz activation. As Fz is activated more in the equatorial cell than the polar cell, Ral is enriched in the equatorial cell. Ral activity represses ligand-independent Notch activation, and thus biases the equatorial cell to become R3. One way in which ligand-independent Notch activation occurs is an accident when normal Notch trafficking is disrupted. Notch receptor undergoes endocytosis and endosomal trafficking continually and mutations that block trafficking of late endosomes to the lysosome block Notch degradation and result in endosomal accumulation of Notch and ligand-independent activation. One possibility is that the endosomal environment promotes production of Nicd by Presenilin cleavage. Ligand-independent Notch activation may also occur normally in the lysosomal membrane. Ral GTPase activity might block ligand-independent Notch activation by regulating Notch trafficking to the lysosome, or by inhibiting another process, such as endosomal acidification, Nicd production or nuclear translocation. The punctate appearance of Ral protein suggests the possibility that Ral may play a role in endosomal trafficking. Although further experiments are required to determine the precise mechanism of Ral function, this study has shown that Ral, a protein that prevents ligand-independent Notch activation, is a target for regulation during pattern formation. Fz/PCP signaling upregulates Ral expression to ensure that ligand-independent Notch activation does not tip the scales in favor of pre-R3 becoming the signal receiving cell. Moreover, this study has shown that prevention of ligand-independent Notch activation is not simply a constitutive process, but one that is modulated to ensure specific developmental outcomes (Cho, 2011).


REFERENCES

Search PubMed for articles about Drosophila Ral

Agapova, L. S., et al. (2004). Activation of Ras-Ral pathway attenuates p53-independent DNA damage G2 checkpoint. J. Biol. Chem. 279: 36382-36389. PubMed ID: 15208305

Asha, H., et al. (1999). The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 18: 605-615. PubMed ID: 9927420

Balakireva, M., et al. (2006). The Ral/exocyst effector complex counters c-Jun N-terminal kinase-dependent apoptosis in Drosophila melanogaster. Mol Cell Biol. 26(23): 8953-63. PubMed ID: 17000765

Beller, M., Blanke, S., Brentrup, D. and Jäckle, H. (2002). Identification and expression of Ima, a novel Ral-interacting Drosophila protein. Mech. Dev. 119 Suppl 1: S253-60. PubMed ID: 14516694

Blanke, S. and Jäckle, H. (2006). Novel guanine nucleotide exchange factor GEFmeso of Drosophila melanogaster interacts with Ral and Rho GTPase Cdc42. FASEB J. 20(6): 683-91. PubMed ID: 16581976

Cantor, S. B., Urano, T. and Feig, L. A. (1995). Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases. Mol. Cell. Biol. 15: 4578-4584. PubMed ID: 7623849

Chien, Y. and White, M. A. (2003). RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival. EMBO Rep. 4: 800-806. PubMed ID: 12856001

Cho, B. and Fischer, J. A. (2011). Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation. Development 138(7): 1349-59. PubMed ID: 21350007

de Ruiter, N. D., et al. (2000). Ras-dependent regulation of c-Jun phosphorylation is mediated by the Ral guanine nucleotide exchange factor-Ral pathway. Mol. Cell. Biol. 20: 8480-8488. PubMed ID: 11046144

Essers, M. A., et al. (2004). FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. EMBO J. 23: 4802-4812. PubMed ID: 15538382

Ghiglione, C., Devergne, O., Cerezo, D. and Noselli, S. (2008). Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles. EMBO Rep. 9(7): 676-82. PubMed ID: 18552769

Gildea, J. J., Harding, M. A., Seraj, M. J., Gulding, K. M. and Theodorescu, D. (2002). The role of Ral A in epidermal growth factor receptor-regulated cell motility. Cancer Res. 62: 982-985. PubMed ID: 11861368

Goi, T., et al. (2000). An EGF receptor/Ral-GTPase signaling cascade regulates c-Src activity and substrate specificity. EMBO J. 19: 623-630. PubMed ID: 10675331

Gonzalez-Garcia, A., et al. (2005). RalGDS is required for tumor formation in a model of skin carcinogenesis. Cancer Cell 7: 219-226. PubMed ID: 15766660

Hamad, N. M., et al. (2002). Distinct requirements for Ras oncogenesis in human versus mouse cells. Genes Dev. 16: 2045-2057. PubMed ID: 12183360

Henry, D. O., et al. (2000). Ral GTPases contribute to regulation of cyclin D1 through activation of NF-B. Mol. Cell. Biol. 20: 8084-8092. PubMed ID: 11027278

Ikeda, M., et al. (1998). Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral. J. Biol. Chem. 273: 814-821. PubMed ID: 9422736

Jullien-Flores, V., Dorseuil, O., Romero, F., Letourneur, F., Saragosti, S., Berger, R., Tavitian, A., Gacon, G. and Camonis, J. H. (1995). Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity. J. Biol. Chem. 270: 22473-22477. PubMed ID: 7673236

Jullien-Flores, V., et al. (2000). RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis. J. Cell Sci. 113: 2837-2844. PubMed ID: 10910768

Kennedy, N. J., et al. (2003). Suppression of Ras-stimulated transformation by the JNK signal transduction pathway. Genes Dev. 17: 629-637. PubMed ID: 12629045

Kops, G. J., et al. (1999). Direct control of the forkhead transcription factor AFX by protein kinase B. Nature 398: 630-634. PubMed ID: 10217147

Mirey, G., et al. (2003). A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways. Mol. Cell Biol. 23(3): 1112-24. PubMed ID: 12529414

Nakashima, S., et al. (1999). Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors. EMBO J. 18(13): 3629-42. PubMed ID: 10393179

Ohta, Y., Suzuki, N., Nakamura, S., Hartwig, J. H. and Stossel, T. P. (1999). The small GTPase RalA targets filamin to induce filopodia. Proc. Natl. Acad. Sci. 96: 2122-2128. PubMed ID: 10051605

Park, S. H. and Weinberg, R. A. (1995). A putative effector of Ral has homology to Rho/Rac GTPase activating proteins. Oncogene 11: 2349-2355. PubMed ID: 8570186

Rangarajan, A., et al. (2004). Species- and cell-type-specific requirements for cellular transformation. Cancer Cell 6: 171-183. PubMed ID: 15324700

Sawamoto, K., et al. (1999a). Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development. Oncogene 18(11): 1967-74. PubMed ID: 10208418

Sawamoto, K., et al. (1999b). The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH(2)-terminal kinase pathway. J. Cell Biol. 146(2): 361-72. PubMed ID: 10427090

Suzuki, J., Yamazaki, Y., Li, G., Kaziro, Y. and Koide, H. (2000) Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts. Mol. Cell. Biol. 20: 4658-4665. PubMed ID: 10848592

Tian, X., et al. (2002). PDK1 mediates growth factor-induced Ral-GEF activation by a kinase-independent mechanism. EMBO J. 21: 1327-1338. PubMed ID: 11889038

Urano, T., Emkey, R. and Feig, L. A. (1996). Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. EMBO J. 15: 810-816. PubMed ID: 8631302

White, M. A., et al. (1996). A role for Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation. J. Biol. Chem. 271: 16439-16442. PubMed ID: 8663585

Wolthuis, R. M. and Bos, J. L. (1999). Ras caught in another affair: the exchange factors for Ral. Curr. Opin. Genet. Dev. 9: 112-117. PubMed ID: 10072355


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date revised: 20 March 2012

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