RalGDS family members couple Ras to Ral signalling and that's not all

Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.     

Rho: a connection between membrane receptor signalling and the cytoskeleton

The Rho family of GTP-binding proteins has yielded fresh insights into cell signalling in relation to motility, shape and the control of the actin cytoskeleton. Rho itself is probably near the top of several diverse signalling cascades and has been implicated in cell adhesion, actin filament organization, control of mitogen-activated protein kinase pathways and phospholipid synthesis and turnover. As a member of the Ras superfamily, Rho is regulated by GDP-GTP exchange factors (GEFs) that have homology to the dbl oncogene, and by GTPase-activating proteins (GAPs). These proteins regulate the nucleotide (GDP or GTP) bound to Rho, thus determining the activity of Rho and the interactions of Rho with many of its downstream targets. In the past year, many new targets of Rho have been identified, which hopefully will uncover molecular connections among the diverse downstream effects of Rho activation.     

Monoterpene regulation of Ras and Ras-related protein expression

Monoterpenes, derived primarily from plants, are products of the isoprenoid biosynthetic pathway and function as chemical messengers with diverse functions. The biochemical bases for these activities are largely undefined. The Ras small GTPase superfamily of proteins consists of isoprenylated proteins that play key roles in signal transduction pathways known to regulate diverse cellular functions. In these studies, we have examined the effects of the monoterpenes on expression of Ras and Ras-related proteins, in the absence and presence of mevalonate depletion. Although prior studies have suggested that monoterpenes inhibit isoprenyl transferases, our studies clearly show that select monoterpenes inhibit up-regulation of Ras and the Ras-related proteins. A structure-activity relationship model for these effects was defined. The ability of monoterpenes to regulate the expression of the Ras-related proteins was found to be independent of effects on cell proliferation or total cellular protein synthesis/degradation. This regulatory function of monoterpenes suggests a role for these plant-derived compounds in altering signal transduction elements.     

RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation

Individual tumour cells move in three-dimensional environments with either a rounded or an elongated 'mesenchymal' morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded/amoeboid movement engages specific Cdc42 and Rho signalling pathways. In siRNA screens targeting the genes encoding guanine nucleotide exchange factors (GEFs), we found that the Ras GEF RasGRF2 regulates conversion between elongated- and rounded-type movement. RasGRF2 suppresses rounded movement by inhibiting the activation of Cdc42 independently of its capacity to activate Ras. RasGRF2 and RasGRF1 directly bind to Cdc42, outcompeting Cdc42 GEFs, thereby preventing Cdc42 activation. By this mechanism, RasGRFs regulate other Cdc42-mediated cellular processes such as the formation of actin spikes, transformation and invasion in vitro and in vivo. These results demonstrate a role for RasGRF GEFs as negative regulators of Cdc42 activation.     

GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.     

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.     

Activation of JNK by Epac is independent of its activity as a Rap guanine nucleotide exchanger

Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac.     

Ras and Rap1: two highly related small GTPases with distinct function

The Ras-like family of small GTPases includes, among others, Ras, Rap1, R-ras, and Ral. The family is characterized by similarities in the effector domain. While the function of Ras is, at least in part, elucidated, little is known about other members of the family. Currently, much attention is focused on the small GTPase Rap1. Initially, this member was identified as a transformation suppressor protein able to revert the morphological phenotype of Ras-transformed fibroblasts. This has led to the hypothesis that Rap1 antagonizes Ras by interfering in Ras effector function. Recent analysis revealed that Rap1 is activated rapidly in response to activation of a variety of receptors. Rap1 activation is mediated by several second messengers, including calcium, diacylglycerol, and cAMP. Guanine nucleotide exchange factors (GEFs) have been identified that mediate these effects. The most interesting GEF is Epac, an exchange protein directly activated by cAMP, thus representing a novel cAMP-induced, protein kinase A-independent pathway. Furthermore, Rap1 is inactivated by specific GTPase-activating proteins (GAPs), one of which is regulated through an interaction with Galphai. While Ras and Rap1 may share some effector pathways, evidence is accumulating that Ras and Rap1 each regulate unique cellular processes in response to various extracellular ligands. For Rap1 these functions may include the control of cell morphology.     

Regulation of small GTPases at epithelial cell-cell junctions

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.     

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.     

Regulation of small GTPases at epithelial cell-cell junctions

Abstract

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.     

Direct interaction of p21-activated kinase 4 with PDZ-RhoGEF, a G protein-linked Rho guanine exchange factor

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.     

GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila

Typical members of the Ras superfamily of small monomeric GTP-binding proteins function as regulators of diverse processes by cycling between biologically active GTP- and inactive GDP-bound conformations. Proteins that control this cycling include guanine nucleotide exchange factors or GEFs, which activate Ras superfamily members by catalyzing GTP for GDP exchange, and GTPase activating proteins or GAPs, which accelerate the low intrinsic GTP hydrolysis rate of typical Ras superfamily members, thus causing their inactivation. Two among the latter class of proteins have been implicated in common genetic disorders associated with an increased cancer risk, neurofibromatosis-1, and tuberous sclerosis. To facilitate genetic analysis, I surveyed Drosophila and human sequence databases for genes predicting proteins related to GAPs for Ras superfamily members. Remarkably, close to 0.5% of genes in both species (173 human and 64 Drosophila genes) predict proteins related to GAPs for Arf, Rab, Ran, Rap, Ras, Rho, and Sar family GTPases. Information on these genes has been entered into a pair of relational databases, which can be used to identify evolutionary conserved proteins that are likely to serve basic biological functions, and which can be updated when definitive information on the coding potential of both genomes becomes available.     

Rho GAPs and GEFs

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.     

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.     

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 drives melanoma cell plasticity

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.     

Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral

Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.     

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.