The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer

Ect2 is an oncogene in multiple human cancers. Ect2 is aberrantly overexpressed in multiple human tumor types, often as a result of targeted amplification of the ECT2 gene as part of the 3q26 amplicon. Ect2 is important for proliferation, migration and invasion of various types of cancer cells in vitro, and for NSCLC tumorigenicity in vivo. The role of Ect2 in cellular transformation is distinct from its physiologic role in cytokinesis, and many tumor cells appear to have evolved Ect2-independent cytokinesis mechanisms. In NSCLC cells, the ability of Ect2 to support transformation is linked to its mislocalization to the cytoplasm and activation of a Rac1-Pak-Mek1,2-Erk1,2 signaling axis that is regulated through its binding to the oncogenic PKCι/Par6α complex (Fig. 4). Therefore, Ect2 and PKCι are genetically linked due to their frequent co-amplification as part of the 3q26 amplicon, and functionally and biochemically linked through formation of an oncogenic PKCι-Par6-Ect2 complex that drives transformation. Further experiments will be required to determine if Ect2 and PKCι are similarly linked in other tumors, particularly those harboring 3q26 amplification. In addition, further work is needed to elucidate the molecular mechanisms that regulate the formation, dynamics and activity of the oncogenic PKCι-Par6α-Ect2 complex. These studies hold the promise of identifying novel therapeutic approaches to cancer treatment based on inhibiting Ect2 function in cancer cells.     

The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage

Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.     

Basis for signaling specificity difference between Sos and Ras-GRF guanine nucleotide exchange factors

Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828-838 in Sos1 and 1057-1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91-103 in Ha-Ras and 117-129 in R-Ras), which interacts instead primarily with helix K (aa 1002-1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.     

Cdc42 and the guanine nucleotide exchange factors Ect2 and trio mediate Fn14-induced migration and invasion of glioblastoma cells

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor-inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14-directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy.     

Molecular basis for the substrate specificity of plant guanine nucleotide exchange factors for ROP

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.     

Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity

Arf6 is an isoform of Arf that localizes at the periphery of the cell where it has an essential role in endocytotic pathways. Its function does not overlap with that of Arf1, although the two proteins share approximately 70% sequence identity and they have switch regions, whose conformation depends on the nature of the guanine nucleotide, with almost identical sequences. The crystal structure of Arf6-GDP at 2.3 A shows that it has a conformation similar to that of Arf1-GDP, which cannot bind membranes with high affinity. Significantly, the switch regions of Arf6 deviate by 2-5 A from those of Arf1. These differences are a consequence of the shorter N-terminal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between Arf1 and Arf6 affects the configuration of the nucleotide binding site and thus the nucleotide binding properties of the Arf variant. Altogether, our results provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a model for the nucleotide/membrane cycle of Arf6.     

The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF

The Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e. p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA (Derewenda, U., Oleksy, A., Stevenson, A. S., Korczynska, J., Dauter, Z., Somlyo, A. P., Otlewski, J., Somlyo, A. V., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1955-1965), we conducted extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF. We show that while Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. We find that selectivity for RhoA versus Cdc42 is defined by a small number of interactions.     

Mechanism of epidermal growth factor regulation of Vav2, a guanine nucleotide exchange factor for Rac

Vav2 is a member of the Vav family that serves as a guanine nucleotide exchange factor for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the epidermal growth factor (EGF) receptor; however, the mechanism by which Vav2 is activated in EGF-treated cells is unclear. By the means of an in vitro protein kinase assay, we show here that purified and activated EGF receptor phosphorylates Vav2 exclusively on its N-terminal domain. Furthermore, EGF receptor phosphorylates Vav2 on all three possible phosphorylation sites, Tyr-142, Tyr-159, and Tyr-172. In intact cells we also show that Vav2 associates with the activated EGF receptor in an Src homology 2 domain-dependent manner, with Vav2 Src homology 2 domain binding preferentially to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. Treatment of cells with EGF results in stimulation of exchange activity of Vav2 as measured on Rac; however, the intensity of the exchange activity does not show any correlation with the level of Vav2 tyrosine phosphorylation. Introducing a point mutation into the Vav2 pleckstrin homology domain or treatment of cells with the phosphatidylinositol 3-kinase inhibitor LY294002 prior to EGF stimulation inhibits Vav2 exchange activity. Although phosphorylation mutants of Vav2 can readily induce actin rearrangement in COS7 cells, pleckstrin homology domain mutant does not stimulate membrane ruffling. These results suggest that EGF regulates Vav2 activity basically through phosphatidylinositol 3-kinase activation, whereas tyrosine phosphorylation of Vav2 may rather be necessary for mediating protein-protein interactions.     

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors

The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.     

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B^RSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)₂ complex. Combined with mutational and biochemical data, we show that eIF2B^RSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα₂ homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B^RSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn⁻, Gcd⁻ and VWM/CACH mutations and the evolutionary history of the eIF2B complex.     

C-terminal motif within Sec7 domain regulates guanine nucleotide exchange activity via tuning protein conformation

ADP-ribosylation factors (Arfs) play key roles in controlling membrane traffic and organelle structures. The activation of Arfs from GDP to GTP binding form is triggered by the guanine exchange factors (GEFs). There are six families of Arf-GEFs with a common guanine exchange catalytic domain (Sec7 domain) and various mechanisms of guanine exchange activity regulation. A loop region (loop>J motif) just following the helix J of Sec7 domain was found conserved and important for the catalytic activity regulation of Arf-GEFs. However, the molecular detail of the role the loop>J motif plays has been yet unclear. Here, we studied the catalytic domain of Sec7p, a yeast trans-Golgi network membrane localized Arf-GEFs, and found that the loop>J motif is indispensible for its GEF catalytic activity. Crystallographic, NMR spectrum and mutagenesis studies suggested that the loop>J motif with a key conserved residue Ile1010 modulates the fine conformation of Sec7 domain and thereby regulates its guanine exchange activity.     

The C-terminal basic tail of RhoG assists the guanine nucleotide exchange factor trio in binding to phospholipids

The multidomain protein Trio regulates among others neuronal outgrowth and axonal guidance in vertebrates and invertebrates. Trio contains two Dbl-homology/pleckstrin homology (DH/PH) tandem domains that activate several RhoGTPases. Here, we present the x-ray structure of the N-terminal DH/PH, hereafter TrioN, refined to 1.7-A resolution. We show that the relative orientations of the DH and PH domains of TrioN and free Dbs are similar. However, this relative orientation is dissimilar to Dbs in the Dbs/Cdc42 structure. In vitro nucleotide exchange experiments catalyzed by TrioN show that RhoG is approximately 3x more efficiently exchanged than Rac and support the conclusion that RhoG is likely the downstream target of TrioN. Residues 54 and 69, which are not conserved between the two GTPases, are responsible for this specificity. Dot-blot assay reveals that the TrioN-PH domain does not detectably bind phosphatidylinositol 3,4-bisphosphate, PtdIns(3,4)P(2), or other phospholipids. This finding is supported by our three-dimensional structure and affinity binding experiments. Interestingly, the presence of RhoG but not Rac or a C-terminal-truncated RhoG mutant allows TrioN to bind PtdIns(3,4)P(2) with a micromolar affinity constant. We conclude the variable C-terminal basic tail of RhoG specifically assists the recruitment of the TrioN-PH domain to specific membrane-bound phospholipids. Our data suggest a role for the phosphoinositide 3-kinase, PI 3-kinase, in modulating the Trio/RhoG signaling pathway.     

pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.     

RasGRP Ras guanine nucleotide exchange factors in cancer

RasGRP proteins are activators of Ras and other related small GTPases by the virtue of functioning as guanine nucleotide exchange factors （GEFs）. In vertebrates, four RasGRP family members have been described. RasGRP-1 through -4 share many structural domains but there are also subtle differences between each of the different family members. Whereas SOS RasGEFs are ubiquitously expressed, RasGRP proteins are expressed in distinct patterns, such as in different cells of the hematopoietic system and in the brain. Most studies have concentrated on the role of RasGRP proteins in the development and function of immune cell types because of the predominant RasGRP expression profiles in these cells and the immune phenotypes of mice deficient for Rasgrp genes. However, more recent studies demonstrate that RasGRPs also play an important role in tumorigenesis. Examples are skin- and hematological- cancers but also solid malignancies such as melanoma or prostate cancer. These novel studies bring up many new and unanswered questions related to the molecular mechanism of RasGRP-driven oncogenesis, such as new receptor systems that RasGRP appears to respond to as well as regulatory mechanisms for RasGRP expression that appear to be perturbed in these cancers. Here we will review some of the known aspects of RasGRP biology in lymphocytes and will discuss the exciting new notion that RasGRP Ras exchange factors play a role in oncogenesis downstream of various growth factor receptors.     

The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.     

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.     

Substrate specificity and recognition is conferred by the pleckstrin homology domain of the Dbl family guanine nucleotide exchange factor P-Rex2

Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.     

PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2

The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.     

RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.     

The importance of P-loop and domain movements in EF-Tu for guanine nucleotide exchange

Elongation factor Ts (EF-Ts) is the guanine nucleotide exchange factor for elongation factor Tu (EF-Tu). An important feature of the nucleotide exchange is the structural rearrangement of EF-Tu in the EF-Tu.EF-Ts complex caused by insertion of Phe-81 of EF-Ts between His-84 and His-118 of EF-Tu. In this study, the contribution of His-118 to nucleotide release was studied by pre-steady state kinetic analysis of nucleotide exchange in EF-Tu mutants in which His-118 was replaced by Ala or Glu. Intrinsic as well as EF-Ts-catalyzed release of GDP/GTP was affected by the mutations, resulting in an approximately 10-fold faster spontaneous nucleotide release and a 10-50-fold slower EF-Ts-catalyzed nucleotide release. The effects are attributed to the interference of the mutations with the EF-Ts-induced movements of the P-loop of EF-Tu and changes at the domain 1/3 interface, leading to the release of the beta-phosphate group of GTP/GDP. The K(d) for GTP is increased by more than 40 times when His-118 is replaced with Glu, which may explain the inhibition by His-118 mutations of aminoacyl-tRNA binding to EF-Tu. The mutations had no effect on EF-Tu-dependent delivery of aminoacyl-tRNA to the ribosome.