Annexins as nucleotide-binding proteins: facts and speculations

Annexins are ubiquitous multifunctional Ca2+ and phospholipid-binding proteins whose mechanism of function remains largely unknown. The accumulated in vitro experimental evidence indicates that ATP and GTP are functional ligands for nucleotide-sensitive annexin isoforms. Such nucleotide binding could modulate Ca2+ homeostasis, vesicular transport and/or signal transduction pathways and link them to cellular energy metabolism. Alternatively, since annexins are able to interact with other nucleotide-utilizing proteins, such as various kinases, GTPases and structural proteins, these proteins could influence the guanine nucleotide exchange metabolism and/or control the activity of various G proteins. The nucleotide-binding properties of annexins may affect the development or maintenance of some pathologies and diseases in which changes in physiological concentrations of purine nucleotides or disruption of Ca2+ homeostasis are crucial targets.     

Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

EhGEF3, a novel Dbl family member, regulates EhRacA activation during chemotaxis and capping in Entamoeba histolytica

Rho GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to cytoskeleton. The Rho guanine nucleotide exchange factors (RhoGEFs) have been implicated in direct activation of these GTPases. Here, we describe a novel RhoGEF, denominated EhGEF3 from the parasite Entamoeba histolytica, which encodes a 110 kDa protein containing the domain arrangement of a Dbl homology domain in tandem with a pleckstrin homology domain, the DH domain of EhGEF3 is closely related with the one of the Vav3 protein. Biochemical analysis revealed that EhGEF3 is capable of stimulating nucleotide exchange on the E. histolytica EhRacA and EhRho1 GTPases in vitro, however only a partial GEF activity toward Cdc42 was observed. Conserved residue analysis showed that the N816 and L817 residues are critical for EhGEF3 activity. Cellular studies revealed that EhGEF3 colocalises with EhRacA in the rear of migrating cells, probably regulating the retraction of the uroid and promoting the activation of these GTPase during the chemotactic response toward fibronectin, and that EhGEF3 also regulates EhRacA activation during the capping of cell receptors. These results suggest that EhGEF3 should have a direct role in activating EhRacA, and in bringing the activated GTPase to specific target sites such as the uroid.     

Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family

Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.     

Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism

Guanine nucleotide exchange factors are essential components of the mode of action of GTPases involved in signal transduction. Their fundamental mechanism is generally accepted to derive from stabilization of the nucleotide-free form of GTPases, which is reflected in an increase in the rate of GDP dissociation when such an exchange factor is bound to a GTPase. The known kinetic properties of exchange factors can be explained on the basis of this simple allosteric competitive mechanism. Here, we describe experiments designed to distinguish this mechanism from a newer model, which invokes an active role for the incoming (i.e., displacing) nucleotide, implying the transient formation of a quaternary complex consisting of an exchange factor, a GTPase, and two nucleotides, one which is being displaced while the other stimulates this displacement. We show that for a well-known system (the small GTPase Ras and its exchange factor Cdc25) there is no evidence for an effect of the concentration or the nature (i.e., GDP or GTP) of the displacing nucleotide on the rate constant of GDP release from the Cdc25.Ras.GDP complex, consistent with the simple allosteric competitive model, and in disagreement with the newer suggestion. In addition, we present arguments, which demonstrate how the erroneous conclusions leading to the alternative model were derived.     

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

Monomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.     

Fluorescence approaches for monitoring interactions of Rho GTPases with nucleotides, regulators, and effectors

To understand the manner in which biological macromolecules interact with each other, we need not only structural information, but also details of kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number of GTPases have been identified and each of these in turn interacts with a variety of regulatory and signaling proteins such as GAPs, GEFs, and downstream effectors. There are a number of methods that can be used to characterize the specificity, strength, and stoichiometry of such intermolecular interactions, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This article discusses the use of fluorescence spectroscopic methods, which allows real-time monitoring of ligand- and protein-protein interactions at submicromolar concentrations, and quantification of the kinetic and equilibrium constants. Fluorescently labeled guanine nucleotides serve as fluorescence reporter groups to investigate the interactions of GTPases of the Rho family (e.g., RhoA, Rac1, and Cdc42). We present examples for quantitative characterization of (i) Rac1 x GDP interaction, (ii) Cdc42-interaction with the GTPase binding domain of the Wiskott Aldrich syndrome protein (three alternative approaches), (iii) accelerated nucleotide exchange reaction of RhoA by the catalytic domains of p190RhoGEF, and (iv) intrinsic and stimulated GTP-hydrolysis reaction by the catalytic domain of p50RhoGAP.     

Structural basis for the signaling specificity of RhoG and Rac1 GTPases

RhoG is a new GTPase that has high sequence similarity with members of the Rac subfamily (Rac1, Rac2, and Rac3), including the regions involved in effector recognition and binding. To characterize its biological properties, we have compared the activity of RhoG and Rac1 in a number of experimental systems, including the study of their subcellular localization, oncogenic potential, activation of effectors, and effect on F-actin dynamics. Our study indicates that RhoG and Rac1 share overlapping, but not identical, signal transduction pathways. In contrast to previous results, we also provide evidence that RhoG works in parallel to Rac1 rather than as a Rac1 upstream activator. Using an extensive collection of Rho/Rac1 chimeras and point mutants, we demonstrate that the different biological properties of RhoG and Rac1 can be traced to specific amino acid variations in their switch I, beta2/beta3 hairpin, alpha5 helix, and C-terminal polybasic regions. Taken collectively, our results highlight the complexity of the signal transduction pathways activated by Rho/Rac GTPases and provide insight into the structural determinants that mediate the differential engagement of biological responses by GTPases of very similar structure.     

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.     

Classification and evolution of P-loop GTPases and related ATPases

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.     

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed.     

p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor

GEF-H1 is a guanine nucleotide exchange factor for Rho whose activity is regulated through a cycle of microtubule binding and release. Here we identify a region in the carboxyl terminus of GEF-H1 that is important for suppression of its guanine nucleotide exchange activity by microtubules. This portion of the protein includes a coiled-coil motif, a proline-rich motif that may interact with Src homology 3 domain-containing proteins, and a potential binding site for 14-3-3 proteins. We identify GEF-H1 as a binding target and substrate for p21-activated kinase 1 (PAK1), an effector of Rac and Cdc42 GTPases, using an affinity-based screen and localize a PAK1 phosphorylation site to the inhibitory carboxyl-terminal region of GEF-H1. We show that phosphorylation of GEF-H1 at Ser(885) by PAK1 induces 14-3-3 binding to the exchange factor and relocation of 14-3-3 to microtubules. Phosphorylation of GEF-H1 by PAK may be involved in regulation of GEF-H1 activity and may serve to coordinate Rho-, Rac-, and Cdc42-mediated signaling pathways.     

Intrinsic protein fluorescence assays for GEF,GAP and post-translational modifications of small GTPases

Evidence and arguments are summarized that suggest that intrinsic (tryptophan) protein fluorescence provides an excellent and convenient signal for monitoring both GEF (guanine nucleotide exchange factor) and GAP (GTPase activating protein) activity of a large number of small GTPases. In addition, post-translational modifications of Rab proteins occurring in a region known to be a hot spot for such modifications also lead to fluorescence changes that can be accurately monitored in a time-dependent manner. It is suggested that intrinsic fluorescence should be the first method chosen for monitoring such reactions of tryptophan-containing small GTPases.     

Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.     

Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.     

Signalling mechanisms of RhoGTPase regulation by the heterotrimeric G proteins G12 and G13

G protein-mediated signal transduction can transduce signals from a large variety of extracellular stimuli into cells and is the most widely used mechanism for cell communication at the membrane. The RhoGTPase family has been well established as key regulators of cell growth, differentiation and cell shape changes. Among G protein-mediated signal transduction, G12/13-mediated signalling is one mechanism to regulate RhoGTPase activity in response to extracellular stimuli. The alpha subunits of G12 or G13 have been shown to interact with members of the RH domain containing guanine nucleotide exchange factors for Rho (RH-RhoGEF) family of proteins to directly connect G protein-mediated signalling and RhoGTPase signalling. The G12/13-RH-RhoGEF signalling mechanism is well conserved over species and is involved in critical steps for cell physiology and disease conditions, including embryonic development, oncogenesis and cancer metastasis. In this review, we will summarize current progress on this important signalling mechanism.     

Structural basis of the Rho GTPase signaling

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.     

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.