Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21.

The stimulation of a variety of cell surface receptors promotes the accumulation of the active, GTP-bound form of Ras proteins in cells. This is a critical step in signal transduction because inhibition of Ras activation by anti-Ras antibodies or dominant inhibitory Ras mutants blocks many of the effects of these receptors on cellular function. To reach the active GTP-bound state, Ras proteins must first release bound GDP. This rate-limiting step in GTP binding is thought to be catalysed by a guanine-nucleotide-releasing factor (GRF). Here we report the cloning of complementary DNAs from a rat brain library that encode a approximately 140K GRF for Ras p21 (p140Ras-GRF). Its carboxy-terminal region is similar to that of CDC25, a GRF for Saccharomyces cerevisiae RAS. This portion of Ras-GRF accelerated the release of GDP from RasH and RasN p21 in vitro, but not from the related RalA, or CDC42Hs GTP-binding proteins. A region in the amino-terminal end of Ras-GRF is similar to both the human breakpoint cluster protein, Bcr, and the dbl oncogene product, a guanine-nucleotide-releasing factor for CDC42Hs. An understanding of Ras-GRF function will enhance our knowledge of the many signal transduction pathways mediated by Ras proteins.     

The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.     

A new family of RhoGEFs activates the Rop molecular switch in plants

In plants, the small GTP-binding proteins called Rops work as signalling switches that control growth, development and plant responses to various environmental stimuli. Rop proteins (Rho of plants, Rac-like and AtRac in Arabidopsis thaliana) belong to the Rho family of Ras-related GTP-binding proteins that turn on signalling pathways by switching from a GDP-bound inactive to a GTP-bound active conformation. Activation depends on guanine nucleotide exchange factors (GEFs) that catalyse the otherwise slow GDP dissociation for subsequent GTP binding. Although numerous RhoGEFs exist in animals and yeasts, no Rop-specific GEFs have yet been identified in plants and so Rop activation has remained elusive. Here we describe a new family of RhoGEF proteins that are exclusive to plants. We define a unique domain within these RopGEFs, termed PRONE (plant-specific Rop nucleotide exchanger), which is exclusively active towards members of the Rop subfamily. It increases nucleotide dissociation from Rop more than a thousand-fold and forms a tight complex with nucleotide-free Rop. RopGEFs may represent the missing link in signal transduction from receptor kinases to Rops and their identification has implications for the evolution of the Rho molecular switch.     

EPS8 and E3B1 transduce signals from Ras to Rac.

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.     

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.     

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the beta-phosphates in the gamma-phosphate-binding site.     

Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins

Several human tumour cell lines contain genes that can transform NIH 3T3 cells into malignant cells. Certain genes have been classified as members of the ras oncogene family, namely, Ha-ras, Ki-ras or N-ras. The proteins encoded by the ras family are generally small (Ha-ras, for example, encodes a protein of molecular weight 21,000 named p21), and are associated with the inner surface of the plasma membrane. The only known biochemical property common to all forms of the ras proteins is the ability to bind guanine nucleotides, a property which may be closely related to the transforming ability of ras proteins. A GTP-dependent, apparent autophosphorylation (on threonine 59) activity has been identified only in the case of the v-Ha-ras protein. Although the role of these biochemical activities in the transformation process remains unclear, we have initiated studies to determine the possible biochemical interactions of ras proteins with other membrane components. We report here the evidence that epidermal growth factor enhances the guanine nucleotide binding activity of activated c-Ha-ras or v-Ha-ras p21, and phosphorylation of v-Ha-ras p21, suggesting that some mitogenic growth factors may regulate those activities.     

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.     

细胞周期末期促进复合物亚基CDC16Hs与DNA双链断端修复蛋白Ku80相互作用

CDC16Hs是细胞周期末期促进复合物(APC)的亚基．利用基于LexA的酵母双杂交系统,把它作为诱饵蛋白筛选人胎脑文库,发现它与DNA双链断端修复蛋白Ku80的羧基端相互作用．CDC16Hs和全长Ku80的结合通过pull down实验在体外得到验证．     

Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.

The fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes. The non-clathrin coatomer complex binds reversibly to Golgi membranes in a GTP-controlled cycle. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes, is required for coatomer binding and probably accounts for the control by guanine nucleotide of the coatomer-membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.     

Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B

Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.     

Primary structure of the alpha-subunit of transducin and its relationship to ras proteins

A group of membrane-associated guanine nucleotide binding proteins (G-proteins) are essential for transducing signals generated at cell-surface receptors into changes in cellular function and metabolism. These proteins are a complex of three subunits designated alpha, beta and gamma. The alpha-subunit is responsible for binding guanine nucleotides and seems to be characteristic of each protein. Transducin, a member of this protein family, mediates visual transduction by coupling the signal of photolysed rhodopsin with activation of a cyclic GMP phosphodiesterase. We have now cloned and sequenced the complementary DNA encoding the alpha-subunit of bovine retinal transducin and from this we have deduced the complete amino-acid sequence. The transducin alpha-subunit shares several homologous amino-acid sequences with ras gene products. The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit.     

Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.     

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.     

Small GTP-binding protein associated with Golgi cisternae

Eukaryotic cells seem to use GTP hydrolysis to regulate vesicular traffic in exocytosis and endocytosis. The best evidence for this comes from studies on the yeast Saccharomyces cerevisiae that have identified two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, which control distinct stages of the secretory pathway. In mammalian cells the effects of a non-hydrolysable GTP analogue, GTP-gamma S, on different transport events have suggested that they also have proteins functionally related to yeast Sec4p and Ypt1p. The rab genes have recently been cloned and sequenced for rat and human and their proteins have highly conserved domains in common with Sec4p and Ypt1p (including a putative effector binding site). They are therefore good candidates for GTP-binding proteins involved in intracellular transport in mammalian cells. One of the Rab proteins (Rab1p) is the mammalian counterpart of Ypt1p (ref. 13). Here we report the localization of the protein Rab6p to the Golgi apparatus in several cell types. By immunolabelling and electron microscopy, Rab6p appears to be concentrated predominantly on the medial and trans cisternae and distributed over their entire surface.     

Legionella pneumophila proteins that regulate Rab1 membrane cycling

Rab1 is a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells. The intracellular pathogen Legionella pneumophila subverts Rab1 function to create a vacuole that supports bacterial replication by a mechanism that is not well understood. Here we describe L. pneumophila proteins that control Rab1 activity directly. We show that a region in the DrrA (defect in Rab1 recruitment A) protein required for recruitment of Rab1 to membranes functions as a guanine nucleotide dissociation inhibitor displacement factor. A second region of the DrrA protein stimulated Rab1 activation by functioning as a guanine nucleotide exchange factor. The LepB protein was found to inactivate Rab1 by stimulating GTP hydrolysis, indicating that LepB has GTPase-activating protein activity that regulates removal of Rab proteins from membranes. Thus, L. pneumophila encodes proteins that regulate three distinct biochemical reactions critical for Rab GTPase membrane cycling to redirect Rab1 to the pathogen-occupied vacuole and to control Rab1 function.     

The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency

Enterohaemorrhagic Escherichia coli attaches to the intestine through actin pedestals that are formed when the bacterium injects its protein EspF(U) (also known as TccP) into host cells. EspF(U) potently activates the host WASP (Wiskott-Aldrich syndrome protein) family of actin-nucleating factors, which are normally activated by the GTPase CDC42, among other signalling molecules. Apart from its amino-terminal type III secretion signal, EspF(U) consists of five-and-a-half 47-amino-acid repeats. Here we show that a 17-residue motif within this EspF(U) repeat is sufficient for interaction with N-WASP (also known as WASL). Unlike most pathogen proteins that interface with the cytoskeletal machinery, this motif does not mimic natural upstream activators: instead of mimicking an activated state of CDC42, EspF(U) mimics an autoinhibitory element found within N-WASP. Thus, EspF(U) activates N-WASP by competitively disrupting the autoinhibited state. By mimicking an internal regulatory element and not the natural activator, EspF(U) selectively activates only a precise subset of CDC42-activated processes. Although one repeat is able to stimulate actin polymerization, we show that multiple-repeat fragments have notably increased potency. The activities of these EspF(U) fragments correlate with their ability to coordinate activation of at least two N-WASP proteins. Thus, this pathogen has used a simple autoinhibitory fragment as a component to build a highly effective actin polymerization machine.