The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
John Sondek - One of the best experts on this subject based on the ideXlab platform.
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GEF means go: Turning on Rho GTPases with Guanine Nucleotide-exchange factors
Nature Reviews Molecular Cell Biology, 2005Co-Authors: Kent L Rossman, Channing J Der, John SondekAbstract:Guanine Nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.
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a crystallographic view of interactions between dbs and cdc42 ph domain assisted Guanine Nucleotide exchange
The EMBO Journal, 2002Co-Authors: Kent L Rossman, David K Worthylake, Jason T Snyder, David P Siderovski, Sharon L Campbell, John SondekAbstract:Dbl-related oncoproteins are Guanine Nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting Guanine Nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in Guanine Nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1·Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.
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crystal structure of rac1 in complex with the Guanine Nucleotide exchange region of tiam1
Nature, 2000Co-Authors: David K Worthylake, Kent L Rossman, John SondekAbstract: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.
Gregory G Tall - One of the best experts on this subject based on the ideXlab platform.
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structure of the g protein chaperone and Guanine Nucleotide exchange factor ric 8a bound to gαi1
Nature Communications, 2020Co-Authors: Levi J Mcclelland, Kaiming Zhang, Tungchung Mou, Jake Johnston, Cindee Yateshansen, Celestine J Thomas, Tzanko Doukov, Sarah Triest, Alexandre Wohlkonig, Gregory G TallAbstract:Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of Nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the Nucleotide-free state of Gα. Ric-8A is Guanine Nucleotide exchange factor (GEF) that also acts as a folding chaperone for its Gα subunit. Here, the authors present the structure of Ric-8A bound to Nucleotide-free Gαi1, revealing the mechanism by which Ric-8A exerts both GEF and chaperone activity.
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mammalian ric 8a synembryn is a heterotrimeric gα protein Guanine Nucleotide exchange factor
Journal of Biological Chemistry, 2003Co-Authors: Gregory G Tall, Andrejs M Krumins, Alfred G GilmanAbstract:The activation of heterotrimeric G proteins is accomplished primarily by the Guanine Nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor Guanine Nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Gαo and Gαs as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent Guanine Nucleotide exchange factor for a subset of Gα proteins including Gαq, Gαi1, and Gαo, but not Gαs. The mechanism of Ric-8A-mediated Guanine Nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Gα proteins, stimulates release of GDP, and forms a stable Nucleotide-free transition state complex with the Gα protein; this complex dissociates upon binding of GTP to Gα.
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ras activated endocytosis is mediated by the rab5 Guanine Nucleotide exchange activity of rin1
Developmental Cell, 2001Co-Authors: Gregory G Tall, Alejandro M Barbieri, Philip D Stahl, Bruce F HorazdovskyAbstract:RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab Guanine Nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 Guanine Nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 Nucleotide exchange activity of RIN1.
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vps9p is a Guanine Nucleotide exchange factor involved in vesicle mediated vacuolar protein transport
Journal of Biological Chemistry, 1999Co-Authors: Hiroko Hama, Gregory G Tall, Bruce F HorazdovskyAbstract:Abstract Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants,VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate Guanine Nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic Guanine Nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel Guanine Nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport.
Alfred Wittinghofer - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterisation of tctp questions its function as a Guanine Nucleotide exchange factor for rheb
FEBS Letters, 2008Co-Authors: Holger Rehmann, Carsten Berghaus, Marc Bruning, Melanie Schwarten, Hugo Stocker, Raphael Stoll, Fried J T Zwartkruis, K. Köhler, Alfred WittinghoferAbstract:Abstract Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a Guanine Nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a Guanine Nucleotide exchange factor for Rheb. Structured summary MINT- 6741806 : RAP1B (uniprotkb: P61224 ) physically interacts (MI: 0218 ) with Epac1 (uniprotkb: O95398 ) by anti tag coimmunoprecipitation (MI: 0007 )
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the Guanine Nucleotide binding switch in three dimensions
Science, 2001Co-Authors: Ingrid R Vetter, Alfred WittinghoferAbstract:Guanine Nucleotide-binding proteins regulate a variety of processes, including sensual perception, protein synthesis, various transport processes, and cell growth and differentiation. They act as molecular switches and timers that cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Recent structural studies show that the switch apparatus itself is a conserved fundamental module but that its regulators and effectors are quite diverse in their structures and modes of interaction. Here we will try to define some underlying principles.
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epac is a rap1 Guanine Nucleotide exchange factor directly activated by cyclic amp
Nature, 1998Co-Authors: J De Rooij, Alfred Wittinghofer, Fried J T Zwartkruis, M H G Verheijen, Robbert H Cool, Sebastian M B Nijman, Johannes L. BosAbstract:Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the Guanine Nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a Guanine-Nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.
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kinetic analysis by fluorescence of the interaction between ras and the catalytic domain of the Guanine Nucleotide exchange factor cdc25mm
Biochemistry, 1998Co-Authors: Christian Lenzen, Robbert H Cool, Heino Prinz, Jurgen Kuhlmann, Alfred WittinghoferAbstract:Guanine Nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, Guanine Nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and Nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543−12552]. The dissociation constant for the binary complex between Nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25Mm285, was 4.6 nM, i.e., a 500-fold lower affinity than the Ras·GDP interaction. The affinities defining the ternary complex Ras·Nucleotide·Cdc25Mm285 are several orders of magnitude lower. The maximum acceleration by Cdc25Mm285 of the GDP dissociation from Ras was more than 105-fold. Kinetic measurements of the association of Nucleotide to Nucleotide-free Ras and to the binary complex Ras· Cdc25Mm285 show that these react...
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differential interaction of the ras family gtp binding proteins h ras rap1a and r ras with the putative effector molecules raf kinase and ral Guanine Nucleotide exchange factor
Journal of Biological Chemistry, 1996Co-Authors: Christian Herrmann, Gudrun Horn, Marcel Spaargaren, Alfred WittinghoferAbstract:Abstract The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral Guanine Nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H-Ras binds with high affinity (K = 20 nM) to Raf-RBD and with low affinity (K = 1 ÂμM) to RGF-RBD, Rap1A shows the opposite behavior. The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF-RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of Guanine Nucleotide, which is the basis for the quantitative measurements in this work. Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-Guanine Nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras.
Martha Vaughan - One of the best experts on this subject based on the ideXlab platform.
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arf Guanine Nucleotide exchange factors big1 and big2 regulate nonmuscle myosin iia activity by anchoring myosin phosphatase complex
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Joel Moss, Martha VaughanAbstract:Brefeldin A-inhibited Guanine Nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf Guanine Nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells.
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the brefeldin a inhibited Guanine Nucleotide exchange protein big2 regulates the constitutive release of tnfr1 exosome like vesicles
Journal of Biological Chemistry, 2007Co-Authors: Aminul Islam, Joel Moss, Martha Vaughan, Xiaoyan Shen, Toyoko Hiroi, Stewart J LevineAbstract:Abstract The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by Guanine Nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited Guanine Nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1β-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.
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interaction of big2 a brefeldin a inhibited Guanine Nucleotide exchange protein with exocyst protein exo70
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kaifeng Xu, Joel Moss, Xiaoyan Shen, Hewang Li, Gustavo Pachecorodriguez, Martha VaughanAbstract:Guanine Nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited Guanine Nucleotide-exchange proteins, BIG1 and BIG2, are important activators of ADP-ribosylation factors for vesicular trafficking. To identify proteins that interact with BIG2, we used cDNA constructs encoding BIG2 sequences in a yeast two-hybrid screen of a human heart library. Clone p2-5-3, encoding a form of human exocyst protein Exo70, interacted with BIG2 amino acids 1–643 and 1–832, but not 644–832, which was confirmed by coimmunoprecipitation of in vitro-translated BIG2 N-terminal segments and 2-5-3. By immunofluorescence microscopy, endogenous BIG2 and Exo70 in HepG2 cells were visualized at Golgi membranes and apparently at the microtubule-organizing center (MTOC). Both were identified in purified centrosomes. Immunoreactive Exo70 and BIG2 partially or completely overlapped with γ-tubulin at the MTOC in cells inspected by confocal microscopy. In cells incubated with brefeldin A, most of the BIG2, Exo70, and trans-Golgi protein p230 were widely dispersed from their perinuclear concentrations, but small amounts always remained, apparently at the MTOC. After disruption of microtubules with nocodazole, BIG2 and Exo70 were widely distributed in cells and remained only partially colocalized with p230, BIG2 more so than Exo70. We conclude that in HepG2 cells BIG2 and Exo70 interact in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane, consistent with a functional association in both early and late stages of vesicular trafficking.
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purification and cloning of a brefeldin a inhibited Guanine Nucleotide exchange protein for adp ribosylation factors
Journal of Biological Chemistry, 1999Co-Authors: Akira Togawa, Joel Moss, Naoko Morinaga, Masahito Ogasawara, Martha VaughanAbstract:Abstract Activation of ADP-ribosylation factors (ARFs), ∼20-kDa Guanine Nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on Guanine Nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ∼200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ∼74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ∼202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 andBIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5′-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.
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structural basis for the inhibitory effect of brefeldin a on Guanine Nucleotide exchange proteins for adp ribosylation factors
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Makoto Sata, Joel Moss, Martha VaughanAbstract:Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are ≈20-kDa GTPases that are active with bound GTP and inactive with GDP bound. Conversion of ARF-GDP to ARF-GTP is regulated by Guanine Nucleotide-exchange proteins. All known ARF Guanine Nucleotide-exchange proteins contain a Sec7 domain of ≈200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA). To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7). Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965–Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect. As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity.
Kent L Rossman - One of the best experts on this subject based on the ideXlab platform.
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GEF means go: Turning on Rho GTPases with Guanine Nucleotide-exchange factors
Nature Reviews Molecular Cell Biology, 2005Co-Authors: Kent L Rossman, Channing J Der, John SondekAbstract:Guanine Nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.
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a crystallographic view of interactions between dbs and cdc42 ph domain assisted Guanine Nucleotide exchange
The EMBO Journal, 2002Co-Authors: Kent L Rossman, David K Worthylake, Jason T Snyder, David P Siderovski, Sharon L Campbell, John SondekAbstract:Dbl-related oncoproteins are Guanine Nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting Guanine Nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in Guanine Nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1·Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.
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crystal structure of rac1 in complex with the Guanine Nucleotide exchange region of tiam1
Nature, 2000Co-Authors: David K Worthylake, Kent L Rossman, John SondekAbstract: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.
