Dictyostelium: an ideal organism for genetic dissection of Ras signalling networks

Signalling pathways based on the small GTPase Ras regulate a multitude of cellular events in eukaryotic cells. Dictyostelium expresses a large and varied family of Ras proteins. It also uses a range of known Ras regulators, in particular RasGEFs, and effectors. The genetic tractability of Dictyostelium, together with the wide range of Ras proteins and regulators, make it an ideal model for the genetic dissection of Ras pathways. This review highlights the recent advances in our understanding of Ras function in Dictyostelium, and considers the implications of these findings for our understanding of eukaryotic signal transduction.     

Redox regulation of Ran GTPase

Ran, a small Ras-like GTP-binding nuclear protein, plays a key role in modulation of various cellular signaling events including the cell cycle. This study shows that a cellular redox agent (nitrogen dioxide) facilitates Ran guanine nucleotide dissociation, and identifies a unique Ran redox architecture involved in that process. Sequence analysis suggests that Dexras1 and Rhes GTPases also possess the Ran redox architecture. As Ran releases an intact nucleotide, the redox regulation mechanism of Ran is likely to differ from the radical-based guanine nucleotide modification mechanism suggested for Ras and Rho GTPases. These results provide a mechanistic reason for the previously observed oxidative stress-induced perturbation of the Ran-mediated nuclear import, and suggest that oxidative stress could be a factor in the regulation of cell signal transduction pathways associated with Ran.     

A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development

BACKGROUND: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins. RESULTS: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins. CONCLUSIONS: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.     

The small nuclear GTPase Ran: How much does it run?

Ran is one of the most abundant and best conserved of the small GTP binding and hydrolyzing proteins of eukaryotes. It is located predominantly in cell nuclei. Ran is a member of the Ras family of GTPases, which includes the Ras and Ras-like proteins that regulate cell growth and division, the Rho and Rac proteins that regulate cytoskeletal organization and the Rab proteins that regulate vesicular sorting. Ran differs most obviously from other members of the Ras family in both its nuclear localization, and its lack of sites required for post-translational lipid modification. Ran is, however, similar to other Ras family members in requiring a specific guanine nucleotide exchange factor (GEF) and a specific GTPase activating protein (GAP) as stimulators of overall GTPase activity. In this review, the multiple cellular functions of Ran are evaluated with respect to its known biochemistry and molecular interactions.     

Small GTPases take the stage

Small GTPases are molecular switches that have been adopted to control many eukaryotic cell functions. Starting with the study of the protooncogene Ras in the early 1980s, detailed pathways have been uncovered upstream and downstream of Ras-related GTP binding proteins. Nonetheless, novel members have been discovered at a pace that has outstripped cell biologists, and thus much remains to be established regarding newer family members. Undiscovered functions are still being uncovered for "established" small GTPases such as Ras, Rho, and Ran. The topics covered at this meeting indeed demonstrate that Ras proteins are at the heart of cellular dynamics.     

Rho GTPase signaling in Dictyostelium discoideum: insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.     

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.     

Spatial Segregation of Ras Signaling—New Evidence from Fission Yeast

The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question—how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras.     

Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains

Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1.     

The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei

Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac-like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co-ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25 degrees C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37 degrees C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA-independent activities. CflA (Cdc42) and CflB (Rac) co-ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).     

Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana

Rho GTPases regulate a number of important cellular functions in eukaryotes, such as organization of the cytoskeleton, stress-induced signal transduction, cell death, cell growth, and differentiation. We have conducted an extensive screening, characterization, and analysis of genes belonging to the Ras superfamily of GTPases in land plants (embryophyta) and found that the Rho family is composed mainly of proteins with homology to RAC-like proteins in terrestrial plants. Here we present the genomic and cDNA sequences of the RAC gene family from the plant Arabidopsis thaliana. On the basis of amino acid alignments and genomic structure comparison of the corresponding genes, the 11 encoded AtRAC proteins can be divided into two distinct groups of which one group apparently has evolved only in vascular plants. Our phylogenetic analysis suggests that the plant RAC genes underwent a rapid evolution and diversification prior to the emergence of the embryophyta, creating a group that is distinct from rac/cdc42 genes in other eukaryotes. In embryophyta, RAC genes have later undergone an expansion through numerous large gene duplications. Five of these RAC duplications in Arabidopsis thaliana are reported here. We also present an hypothesis suggesting that the characteristic RAC proteins in higher plants have evolved to compensate the loss of RAS proteins.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.     

The RasGEF FgCdc25 regulates fungal development and virulence in Fusarium graminearum via cAMP and MAPK signalling pathways

Ras GTPases act as molecular switches to control various cellular processes by coupling integrated signals in eukaryotes. Activities of Ras GTPases are triggered by Ras GTPase guanine nucleotide exchange factors (RasGEFs) in general, whereas the role of RasGEF in plant pathogenic fungi is largely unknown. In this study, we characterized the only RasGEF protein in Fusarium graminearum, FgCdc25, by combining genetic, cytological and phenotypic strategies. FgCdc25 directly interacted with RasGTPase FgRas2, but not FgRas1, to regulate growth and sexual reproduction. Mutation of the FgCDC25 gene resulted in decreased toxisome formation and deoxynivalenol (DON) production, which was largely depended on cAMP signalling. In addition, FgCdc25 indirectly interacted with FgSte11 in FgSte11-Ste7-Gpmk1 cascade, and the ΔFgcdc25 strain totally abolished the formation of infection structures and was nonpathogenic in planta, which was partially recovered by addition of exogenous cAMP. In contrast, FgCdc25 directly interplayed with FgBck1 in FgBck1-MKK1-Mgv1 cascade to negatively control cell wall integrity. Collectively, these results suggest that FgCdc25 modulates cAMP and MAPK signalling pathways and further regulates fungal development, DON production and plant infection in F. graminearum.     

Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR

Directed cell migration and cell polarity are crucial in many facets of biological processes. Cellular motility requires a complex array of signaling pathways, in which orchestrated cross-talk, a feedback loop, and multi-component signaling recur. Almost every signaling molecule requires several regulatory processes to be functionally activated, and a lack of a signaling molecule often leads to chemotaxis defects, suggesting an integral role for each component in the pathway. We outline our current understanding of the signaling event that regulates chemotaxis with an emphasis on recent findings associated with the Ras, PI3K, and target of rapamycin (TOR) pathways and the interplay of these pathways. Ras, PI3K, and TOR are known as key regulators of cellular growth. Deregulation of those pathways is associated with many human diseases, such as cancer, developmental disorders, and immunological deficiency. Recent studies in yeast, mammalian cells, and Dictyostelium discoideum reveal another critical role of Ras, PI3K, and TOR in regulating the actin cytoskeleton, cell polarity, and cellular movement. These findings shed light on the mechanism by which eukaryotic cells maintain cell polarity and directed cell movement, and also demonstrate that multiple steps in the signal transduction pathway coordinately regulate cell motility.     

RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants

RAC/ROP GTPases are a family of plant-specific signaling molecules solely representing the Ras and Rho family of Ras-related G proteins in plants. RAC/ROPs potentially interact with cell surface-associated signal perception apparatus for a broad range of extracellular stimuli, including hormones, pathogen elicitors and abiotic stress, and mediate diverse cellular pathways in response to these signals. They are also known to interact with multiple effectors, affecting cellular and biochemical systems that regulate actin dynamics, reactive oxygen species production, proteolysis, and gene expression. RAC/ROPs are, thus, ideally suited as integrators for multiple signals and as coordinators of diverse cellular pathways to control growth, differentiation, development and defense responses. Recent findings that suggest how RAC/ROP signaling activity is regulated and how functional specificity can be achieved are discussed here.     

Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.     

How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.