GrinchGEF--a novel Rho-specific guanine nucleotide exchange factor

RhoGTPases, which are activated by specific guanine nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a new RhoGEF (GrinchGEF) containing the typical Dbl homology domain, a putative WD40-like domain, and two predicted transmembrane helices. In contrast to most other RhoGEFs, it exhibits no sequence similarities to known pleckstrin homology domains. GrinchGEF mRNA was highly abundant in skeletal muscle and pancreas. Despite the predicted transmembrane domains, subcellular localization studies revealed a cytosolic distribution. In vitro, GrinchGEF induced the GDP/GTP exchange at RhoA, but not at Rac1 or Cdc42. In intact cells, GrinchGEF induced specifically Rho activation and enhanced RhoA-C-specific downstream effects.     

ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.     

The guanine nucleotide exchange factor Tiam1: A Janus-faced molecule in cellular signaling

The Rho family of GTPases consists of several small proteins that have been described as molecular switches, playing important roles in a wide variety of fundamental cellular processes and in human diseases such as cancer. These proteins, active in the GTP conformation and inactive in the GDP form, are in turn regulated by guanine nucleotide exchange factors (GEFs), guanine nucleotide activating proteins (GAPs) and guanine dissociation inhibitors (GDIs). Two decades ago, Tiam1 (T-lymphoma invasion and metastasis) was identified as a GEF specific for Rac1 activation, but also for Cdc42 and in a lesser extent RhoA.Acting principally upstream of Rac1, Tiam1 is mainly involved in the regulation of Rac1 mediated signaling pathways including cytoskeletal activities, cell polarity, endocytosis and membrane trafficking, cell migration, adhesion and invasion, cell growth and survival, metastasis and carcinogenesis. However, given the large number of protein interaction domains found in its structure, it is possible that Tiam1 affects cellular processes in another way than through its GEF activity by interactions with other signaling proteins.Due to its functional diversity, Tiam1 is involved in multiple steps of tumorigenesis.As its name suggests, Tiam1 has been shown to increase T-cell lymphoma invasion and metastasis. It also promotes migration of fibroblasts, neuronal and cancer cells. On the contrary, Tiam1-induced cell adhesion has also been described, as opposed to cell migration. Moreover, studies indicate that Tiam1 is involved in both anti-apoptotic and pro-apoptotic mechanisms.While increasing evidence has demonstrated Tiam1's contribution to tumorigenesis and metastasis, others suggest that Tiam1 could have anti-cancer properties.In the present review, we discuss the current knowledge about the controversial roles of Tiam1 in cellular signaling. In particular, we will focus on Tiam1's regulation, its biological functions and implication in cancer.     

ADP-ribosylation factor/COPI-dependent events at the endoplasmic reticulum-Golgi interface are regulated by the guanine nucleotide exchange factor GBF1

ADP-ribosylation factor (ARF) mediated recruitment of COPI to membranes plays a central role in transport between the endoplasmic reticulum (ER) and the Golgi. The activation of ARFs is mediated by guanine nucleotide exchange factors (GEFs). Although several ARF-GEFs have been identified, the transport steps in which they function are still poorly understood. Here we report that GBF1, a member of the Sec7-domain family of GEFs, is responsible for the regulation of COPI-mediated events at the ER-Golgi interface. We show that GBF1 is essential for the formation, differentiation, and translocation of pre-Golgi intermediates and for the maintenance of Golgi integrity. We also show that the formation of transport-competent ER-to-Golgi intermediates proceeds in two stages: first, a COPI-independent event leads to the formation of an unstable compartment, which is rapidly reabsorbed in the absence of GBF1 activity. Second, the association of GBF1 with this compartment allows COPI recruitment and leads to its maturation into transport intermediates. The recruitment of GBF1 to this compartment is specifically inhibited by brefeldin A. Our findings imply that the continuous recruitment of GBF1 to spatially differentiated membrane domains is required for sustained membrane remodeling that underlies membrane traffic and Golgi biogenesis.     

The isolated catalytic hairpin of the Ras-specific guanine nucleotide exchange factor Cdc25Mm retains nucleotide dissociation activity but has impaired nucleotide exchange activity

Cdc25Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation--final steps in the GEF catalytic cycle--require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.     

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.     

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.     

Ccpg1, a novel scaffold protein that regulates the activity of the Rho guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.     

EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.     

GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors,is localized to the cis-Golgi and involved in membrane association of the COPI coat

Formation of coated carrier vesicles, such as COPI-coated vesicles from the cis -Golgi, is triggered by membrane binding of the GTP-bound form of ADP-ribosylation factors. This process is blocked by brefeldin A, which is an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor. GBF1 is one of the guanine nucleotide-exchange factors for ADP-ribosylation factor and is localized in the Golgi region. In the present study, we have determined the detailed subcellular localization of GBF1. Immunofluorescence microscopy of cells treated with nocodazole or incubated at 15 °C has suggested that GBF1 behaves similarly to proteins recycling between the cis -Golgi and the endoplasmic reticulum. Immunoelectron microscopy has revealed that GBF1 localizes primarily to vesicular and tubular structures apposed to the cis -face of Golgi stacks and minor fractions to the Golgi stacks. GBF1 overexpressed in cells causes recruitment of class I and class II ADP-ribosylation factors onto Golgi membranes. Furthermore, overexpressed GBF1 antagonizes various effects of brefeldin A, such as inhibition of membrane recruitment of ADP-ribosylation factors and the COPI coat, and redistribution of Golgi-resident and itinerant proteins. These observations indicate that GBF1 is involved in the formation of COPI-coated vesicles from the cis -Golgi or the pre-Golgi intermediate compartment through activating ADP-ribosylation factors.     

Effect of diabetes on guanine nucleotide exchange factor activity in skeletal muscle and heart

The present study examined the effects of diabetes and insulin treatment of diabetic rats on the activity of the protein synthesis initiation factor, the guanine nucleotide exchange factor. In extracts from gastrocnemius and psoas muscles from two-day diabetic rats, guanine nucleotide exchange factor activity was reduced to 80% and 67% of control values, respectively. Insulin treatment (2 h) restored guanine nucleotide exchange factor activity to control values in both muscles. In contrast, guanine nucleotide exchange factor activity was unchanged in extracts from either soleus muscle or heart from diabetic rats compared to controls. Also, insulin treatment did not increase guanine nucleotide exchange factor activity in extracts from soleus and heart. The results suggest that the diabetes-induced impairment in peptide-chain initiation in fast-twitch skeletal muscle (i.e. gastrocnemius and psoas) is related to an inhibition of guanine nucleotide exchange factor activity and that slow-twitch muscle is spared from the effect on initiation due to the preservation of guanine nucleotide exchange factor activity.     

Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac

Epac is a cAMP-dependent exchange factor for the small GTP-binding protein Rap. The activity of Epac is inhibited by a direct interaction between the C-terminal helical part of the cAMP-binding domain, called the lid, and the catalytic region, which is released after binding of cAMP. Herein, we show that the activation properties are very sensitive to modifications of the cyclic nucleotide. Some analogues are inhibitory and others are stimulatory; some are characterized by a much higher activation potential than normal cAMP. Mutational analysis of Epac allows insights into a network of interactions between the cyclic nucleotides and Epac. Mutations in the lid region are able to amplify or to attenuate selectively the activation potency of cAMP analogues. The properties of cAMP analogues previously used for the activation of the cAMP responsive protein kinase A and of 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate, an analogue highly selective for activation of Epac were investigated in detail.     

Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1.     

Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in adipocytes

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.     

Very-KIND is a novel nervous system specific guanine nucleotide exchange factor for Ras GTPases

The kinase non-catalytic c-lobe domain (KIND) evolved from the catalytic protein kinase fold into a potential protein interaction module for signalling proteins. Spir family actin organizers and the non-receptor phosphatase type 13 (PTP type 13) encode a KIND domain in the very N-terminal parts of the proteins. Here we report the characterization and cloning of a third member of the KIND protein family, which we have named very-KIND (VKIND) because of its two KIND domains. Like the other members of the protein family, VKIND has a KIND domain at the N-terminus. A second KIND domain is located in the central part of the protein. The C-terminal half encodes a guanine nucleotide exchange factor motif for Ras-like GTPases (RasGEF) and a RasGEF N-terminal module (RasGEFN). There is only one VKIND gene in the mammalian genomes and up to now we have found the gene only in vertebrates. During mouse embryogenesis the VKIND gene was specifically expressed in the developing nervous system. In adult mice Northern hybridizations revealed high expression only in brain. Low expression could be detected in ovary. In situ hybridizations showed a specific expression of VKIND in neuronal cells of the granular and Purkinje cell layers of the cerebellum.     

Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity

Guanine nucleotide exchange factor (GEF) domains of the Dbl family occur in a variety of proteins that include multiple protein-protein and protein-lipid interaction domains. We used an epithelial-derived cell line to investigate the mechanisms by which the two GEF domains of Kalirin, a neuronal Rho GEF, influence morphology. As expected, Kal-GEF1, an efficient GEF for Rac1 and RhoG, induced the formation of lamellipodia resembling those induced by active Rac1. Although Kal-GEF1 activated Rac and Pak, its ability to induce formation of lamellipodia was not blocked by dominant negative Rho GTPases or by catalytically inactive Pak. Consistent with this, a catalytically inactive mutant of Kal-GEF1 induced formation of lamellipodia and activated Pak. Active Pak was required for the GEF-activity independent effect of Kal-GEF1 and the lamellipodia produced were filled with ribs of filamentous actin. Kal-GEF1 and a GEF-dead mutant co-immunoprecipitated with Pak. The interaction of Kal-GEF1 with Pak is indirect and requires the regulatory protein binding domain of Pak. Filamin A, which is known to interact with and activate Pak, binds to both catalytically active and inactive Kal-GEF1, providing a link by which catalytically inactive Kal-GEF1 can activate Pak and induce lamellipodia. Together, our results indicate that Kal-GEF1 induces lamellipodia through activation of Pak, where GEF activity is not required. GEF-activity-independent effects on downstream targets may be a general property of RhoGEFs.     

Nuclear functions of the Arf guanine nucleotide exchange factor BRAG2

BRAG2 is a guanine nucleotide exchange factor for the GTPase Arf6 that cycles between the cytoplasm and nucleus in a CRM1/exportin1-dependent manner. Despite its presence in the nucleus, nuclear functions have not previously been described. Here, we show that depletion of endogenous BRAG2 by RNAi leads to an increased number of Cajal bodies (CBs), and altered structure of nucleoli, as indicated by less focal fibrillarin staining. This result was surprising given that nuclear BRAG2 is diffusely distributed throughout the nucleoplasm and is not concentrated within nucleoli at steady state. However, we found that ectopic expression of the nuclear GTPase PIKE/AGAP2 causes both BRAG2 and the CB marker coilin to accumulate in nucleoli. Neither the GTPase activity of PIKE nor the nucleotide exchange activity of BRAG2 is required for this nucleolar concentration. Increased levels of exogenous BRAG2 in nucleoli result in a redistribution of fibrillarin to the nucleolar periphery, supporting a role for BRAG2 in regulating nucleolar architecture. These observations suggest that, in addition to its role in endocytic regulation at the plasma membrane, BRAG2 also functions within the nucleus.