Small GTPase

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Andreas Niederwanger - One of the best experts on this subject based on the ideXlab platform.

  • serotonin improves glucose metabolism by serotonylation of the Small GTPase rab4 in l6 skeletal muscle cells
    Diabetology & Metabolic Syndrome, 2017
    Co-Authors: R Alzoairy, Michael T Pedrini, Mohammad Imran Khan, Julia Engl, Alexander Tschoner, C F Ebenbichler, Gerhard Gstraunthaler, Karin Salzmann, Rania Bakry, Andreas Niederwanger
    Abstract:

    Serotonin (5-HT) improves insulin sensitivity and glucose metabolism, however, the underlying molecular mechanism has remained elusive. Previous studies suggest that 5-HT can activate intracellular Small GTPases directly by covalent binding, a process termed serotonylation. Activated Small GTPases have been associated with increased GLUT4 translocation to the cell membrane. Therefore, we investigated whether serotonylation of Small GTPases may be involved in improving Insulin sensitivity and glucose metabolism. Using fully differentiated L6 rat skeletal muscle cells, we studied the effect of 5-HT in the absence or presence of insulin on glycogen synthesis, glucose uptake and GLUT4 translocation. To prove our L6 model we additionally performed preliminary experiments in C2C12 murine skeletal muscle cells. Incubation with 5-HT led to an increase in deoxyglucose uptake in a concentration-dependent fashion. Accordingly, GLUT4 translocation to the cell membrane and glycogen content were increased. These effects of 5-HT on Glucose metabolism could be augmented by co-incubation with insulin and blunted by co incubation of 5-HT with monodansylcadaverine, an inhibitor of protein serotonylation. In accordance with this observation, incubation with 5-HT resulted in serotonylation of a protein with a molecular weight of approximately 25 kDa. We identified this protein as the Small GTPase Rab4, the activity of which has been shown to be stimulated by both insulin signalling and serotonylation. Our data suggest that 5-HT elicits its beneficial effects on Glucose metabolism through serotonylation of Rab4, which likely represents the converging point between the insulin and the 5-HT signalling cascades.

  • Serotonin improves glucose metabolism by Serotonylation of the Small GTPase Rab4 in L6 skeletal muscle cells
    Diabetology & Metabolic Syndrome, 2017
    Co-Authors: Ramona Al-zoairy, Michael T Pedrini, Mohammad Imran Khan, Julia Engl, Alexander Tschoner, C F Ebenbichler, Gerhard Gstraunthaler, Karin Salzmann, Rania Bakry, Andreas Niederwanger
    Abstract:

    Background Serotonin (5-HT) improves insulin sensitivity and glucose metabolism, however, the underlying molecular mechanism has remained elusive. Previous studies suggest that 5-HT can activate intracellular Small GTPases directly by covalent binding, a process termed serotonylation. Activated Small GTPases have been associated with increased GLUT4 translocation to the cell membrane. Therefore, we investigated whether serotonylation of Small GTPases may be involved in improving Insulin sensitivity and glucose metabolism. Methods Using fully differentiated L6 rat skeletal muscle cells, we studied the effect of 5-HT in the absence or presence of insulin on glycogen synthesis, glucose uptake and GLUT4 translocation. To prove our L6 model we additionally performed preliminary experiments in C2C12 murine skeletal muscle cells. Results Incubation with 5-HT led to an increase in deoxyglucose uptake in a concentration-dependent fashion. Accordingly, GLUT4 translocation to the cell membrane and glycogen content were increased. These effects of 5-HT on Glucose metabolism could be augmented by co-incubation with insulin and blunted by co incubation of 5-HT with monodansylcadaverine, an inhibitor of protein serotonylation. In accordance with this observation, incubation with 5-HT resulted in serotonylation of a protein with a molecular weight of approximately 25 kDa. We identified this protein as the Small GTPase Rab4, the activity of which has been shown to be stimulated by both insulin signalling and serotonylation. Conclusion Our data suggest that 5-HT elicits its beneficial effects on Glucose metabolism through serotonylation of Rab4, which likely represents the converging point between the insulin and the 5-HT signalling cascades.

Fried J T Zwartkruis - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the Small GTPase Rheb by amino acids
    Oncogene, 2006
    Co-Authors: Marta Roccio, Johannes L. Bos, Fried J T Zwartkruis
    Abstract:

    The mTOR/S6K/4E-BP1 pathway integrates extracellular signals derived from growth factors, and intracellular signals, determined by the availability of nutrients like amino acids and glucose. Activation of this pathway requires inhibition of the tumor suppressor complex TSC1/2. TSC2 is a GTPase-activating protein for the Small GTPase Ras homologue enriched in brain (Rheb), GTP loading of which activates mTOR by a yet unidentified mechanism. The level at which this pathway senses the availability of amino acids is unknown but is suggested to be at the level of TSC2. Here, we show that amino-acid depletion completely blocks insulin- and TPA-induced Rheb activation. This indicates that amino-acid sensing occurs upstream of Rheb. Despite this, amino-acid depletion can still inhibit mTOR/S6 kinase signaling in TSC2-/- fibroblasts. Since under these conditions Rheb-GTP levels remain high, a second level of amino-acid sensing exists, affecting mTOR activity in a Rheb-independent fashion.

  • the Small GTPase rap1 mediates cd31 induced integrin adhesion
    Journal of Cell Biology, 2000
    Co-Authors: Kris A Reedquist, Fried J T Zwartkruis, Rob M. F. Wolthuis, Ewan A Ross, Elianne A Koop, Yvette Van Kooyk, M Salmon, Christopher D Buckley
    Abstract:

    Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via β1 (VLA-4) and β2 (LFA-1) integrins. We identify the Small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the Small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.

  • Ras-dependent activation of the Small GTPase Ral.
    Current Biology, 1998
    Co-Authors: Rob M. F. Wolthuis, Fried J T Zwartkruis, Tessa C. Moen
    Abstract:

    Abstract The Small GTPase Ral is a Ras-like GTPase [1] that has been implicated in growth-factor-induced and Ras-induced DNA synthesis [2–4], and Ras-induced oncogenic transformation [3,5]. Recently, we and others found that three different Ral guanine nucleotide exchange factors (Ral GEFs) – Ral GDS, Rgl and Rlf – bind specifically to the GTP-bound form of several Ras-like GTPases [6–9]. Although oncogenic Ras is able to activate these Ral GEFs [2,5,10], it is unknown whether growth factors can induce the activation of Ral and, if so, which Small GTPase is involved in this process. Here, we show that stimulation of various growth factor receptors, including receptor tyrosine kinases and serpentine receptors, results in rapid activation of Ral. This activation correlates with the activation of Ras, and dominant-negative Ras completely inhibits Ral activation induced by insulin and epidermal growth factor (EGF). From these results, we conclude that Ral activation is a direct downstream effect of growth-factor-induced Ras activation

Zoe Powis - One of the best experts on this subject based on the ideXlab platform.

  • de novo mutations in the gtp gdp binding region of rala a ras like Small GTPase cause intellectual disability and developmental delay
    PLOS Genetics, 2018
    Co-Authors: Susan M Hiatt, Jeremy W Prokop, Ryne C Ramaker, Andrew A Hardigan, Miroslava Hancarova, Darina Prchalova, Marketa Havlovicova, Jan Prchal, Viktor Stranecky, Zoe Powis
    Abstract:

    Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like Small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10−11) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in Small GTPases.

  • de novo mutations in the gtp gdp binding region of rala a ras like Small GTPase cause intellectual disability and developmental delay
    bioRxiv, 2018
    Co-Authors: Susan M Hiatt, Jeremy W Prokop, Ryne C Ramaker, Andrew A Hardigan, Miroslava Hancarova, Darina Prchalova, Marketa Havlovicova, Jan Prchal, Viktor Stranecky, Zoe Powis
    Abstract:

    Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies, but the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins, and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like Small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p=4.93 x 10-11) than expected from the estimated mutation rate. Further, all de novo variants described here affect conserved residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. We directly assayed GTP hydrolysis and RALA effector-protein binding, and all but one tested variant significantly reduced both activities. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in Small GTPases.

Hideki Sumimoto - One of the best experts on this subject based on the ideXlab platform.

  • direct involvement of the Small GTPase rac in activation of the superoxide producing nadph oxidase nox1
    Journal of Biological Chemistry, 2006
    Co-Authors: Kei Miyano, Noriko Ueno, Ryu Takeya, Hideki Sumimoto
    Abstract:

    Abstract Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22phox at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the Small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.

  • tetratricopeptide repeat tpr motifs of p67 phox participate in interaction with the Small GTPase rac and activation of the phagocyte nadph oxidase
    Journal of Biological Chemistry, 1999
    Co-Authors: Hirofumi Koga, Hiroaki Terasawa, Hiroyuki Nunoi, Koichiro Takeshige, Fuyuhiko Inagaki, Hideki Sumimoto
    Abstract:

    Abstract The Small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67phox, another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67phox N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly α-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo andin vitro. Thus the TPR motifs of p67phox are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.

  • phosphoinositide 3 kinase dependent and independent activation of the Small GTPase rac2 in human neutrophils
    Journal of Biological Chemistry, 1999
    Co-Authors: Takashi Akasaki, Hirofumi Koga, Hideki Sumimoto
    Abstract:

    Abstract The Small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonistsN-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.

Cecilia Bucci - One of the best experts on this subject based on the ideXlab platform.

  • cystinosin the Small GTPase rab11 and the rab7 effector rilp regulate intracellular trafficking of the chaperone mediated autophagy receptor lamp2a
    Journal of Biological Chemistry, 2017
    Co-Authors: Jinzhong Zhang, Cecilia Bucci, Jennifer L Johnson, Mahalakshmi Ramadass, Jing He, Evripidis Gavathiotis, Gennaro Napolitano, Celine J Rocca, William B Kiosses, Ana Maria Cuervo
    Abstract:

    Author(s): Zhang, Jinzhong; Johnson, Jennifer L; He, Jing; Napolitano, Gennaro; Ramadass, Mahalakshmi; Rocca, Celine; Kiosses, William B; Bucci, Cecilia; Xin, Qisheng; Gavathiotis, Evripidis; Cuervo, Ana Maria; Cherqui, Stephanie; Catz, Sergio D | Abstract: The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the Small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with Small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

  • multiple roles of the Small GTPase rab7
    Cells, 2016
    Co-Authors: Flora Guerra, Cecilia Bucci
    Abstract:

    Rab7 is a Small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes.

  • role of the Small GTPase rab7 in the late endocytic pathway
    Journal of Biological Chemistry, 1997
    Co-Authors: Rosalba Vitelli, Mariarosaria Santillo, Daniela Lattero, Mario Chiariello, Maurizio Bifulco, Carmelo B Bruni, Cecilia Bucci
    Abstract:

    Abstract Rab7 is a Small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many Small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

  • the Small GTPase rab5 functions as a regulatory factor in the early endocytic pathway
    Cell, 1992
    Co-Authors: Cecilia Bucci, Robert G. Parton, Ian H Mather, Henk Stunnenberg, Kai Simons, Bernard Hoflack, Marino Zerial
    Abstract:

    Abstract We have investigated the in vivo functional role of rab5, a Small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5ile 133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of Small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a ratelimiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.