Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

Ohga, Nobuyuki; Kikuchi, Akira; Ueda, Takashi; Yamamoto, Juro; Takai, Yoshimi

doi:10.1016/0006-291x(89)91153-4

Cited by 46 publications

(24 citation statements)

References 30 publications

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“…Nevertheless, smg p25A GDI has no significant homology with the Py subunits. On the other hand, we have recently isolated the GDI specific for rhoB p20 from rabbit intestine tissue (19). We have also purified it to near homogeneity from bovine brain cytosol (T. Ueda, A.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

Matsui¹,

Kikuchi²,

Araki³

et al. 1990

Mol. Cell. Biol.

157

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We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the fry subunits of GTP-binding proteins having an afry subunit structure, such as G. and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent. smg p25A was first purified from bovine brain membranes and has been characterized (11). The primary structure of smg p25A has been determined on the basis of the nucleotide sequence of its cDNA (16). The physical and kinetic properties of smg p25A indicate that this protein is a member of a superfamily of ras p21-ras p21-like GTP-binding proteins (G proteins) (for reviews, see references 1 and 27). The counterpart of smg p25A in rats has been reported to be the rab3-encoded protein (34). Although the functions of this G protein have not been clarified, it has been shown that the smg p25A gene is highly expressed in nerve tissues (21,23) and that its mRNA level is increased markedly after differentiation of PC12 cells into neuronlike cells by nerve growth factor or dibutyryl cyclic AMP (23). Our recent immunocytochemical studies have revealed that smg p25A is found not only in neuronal cells and adrenal glands but also in other secretory cells, such as endocrine and/or exocrine cells of the pancreas, parotid gland, and pituitary gland (17; A. Mizoguchi, S. Kim, T. Ueda, A. Kikuchi, H. Yorifuji, N. Hirokawa, and Y. Takai, J. Biol. Chem., in press). Moreover, our results indicate that in brain tissue, smg p25A is found mostly in presynapses, where it is localized mainly in synaptic plasma membranes and partly in synap...

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

Matsui¹,

Kikuchi²,

Araki³

et al. 1990

Mol. Cell. Biol.

157

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“…GTP bound Rac1 associates with the membrane through an isoprenylated C terminus. Upon the inactivation of Rac1 by GAP, RhoGDI, a guanine dissociation inhibitor, dissociates GDP-bound Rac1 from the membrane and sequesters it in a GDP-bound state in the cytosol (30,35,37).…”

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Examination of the Coordinate Effects ofPseudomonas aeruginosaExoS on Rac1

et al. 2005

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Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.Pseudomonas aeruginosa is a ubiquitous, environmentally beneficial bacterium that can adapt to become a highly virulent opportunistic pathogen in compromised individuals. The versatility and pathogenicity of P. aeruginosa are multifactorial and relate to its ability to respond to its environment by the regulated production of a variety of cell-associated and extracellular products. The establishment of infection begins with the adherence of P. aeruginosa to host cells through type IV pili or nonpilus adherence mechanisms (42, 50, 52). After colonization, the organism ensures its survival in the host through the secretion of virulence factors, including exotoxin A (24), hemolysins (40), elastases LasA and LasB (2,20,21), and pigments (53). In addition to secreted virulence factors, P. aeruginosa is able to directly affect eukaryotic cell function through the contact-dependent translocation of effector proteins by the type III secretion system (59). Four P. aeruginosa-type III secretory (TTS) effector proteins have been identified, ExoS, ExoT, ExoU, and ExoY, each affecting eukaryotic cell function differently. Notably, the TTS system is found in both clinical and environmental P. aeruginosa isolates, suggesting an essential role of TTS in P. aeruginosa overall growth and survival (8,9,49). The integral relationship between P. aeruginosa TTS effectors and eukaryotic cell function is also evident in the requirement of eu...

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“…The GDP/GTP exchange and GTPase reactions are regulated by GDP/GTP exchange protein (GEP) and GTPase activating protein, respectively (2,3). There are stimulatory and inhibitory GEPs, named GDP dissociation stimulator (GDS) and GDP dissociation inhibitor, respectively (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). GDSs have been identified for ras p2ls, smg p2ls, and rho p2ls (14)(15)(16)(17)(18)(19)(20), whereas GDP dissociation inhibitors have been identified for rho p2is and smg p25A (9)(10)(11)(12)(13).…”

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A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21.

Mizuno

Kaibuchi

Yamamoto

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

188

142

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A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21 (ras ABSTRACTWe have purified a stimulatory GDP/GTP exchange protein for smg p21A and -B, ras p21-like small GTP-binding proteins (G proteins), cloned its cDNA, and named it GDP dissociation stimulator (smg p21 GDS). We show here that smg p21 GDS is active not only on smg p21A and -B but also on c-Ki-ras p21 and rhoA p21, all of which are post-translationally processed. Furthermore, we show that smig p21 GDS is inactive on the post-translationally unprocessed form of these proteins and on the post-translationally processed form of c-Ha-ras p21 and smg p25A. All of the small G proteins recognized by smg p21 GDS have a cDNA-predicted C-terminal "CAAX" motif (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid) and a polybasic region upstream of this motif. These results suggest that smg p21 GDS is at least active on a group of small G proteins having these unique C-terminal structures. Moreover, they suggest that the C-terminal post-translational processing of these small G proteins, by farnesylation or geranylgeranylation of the C-terminal cysteine residue, removal of amino acids in positions denoted "AAX", and carboxyl methylation of the exposed cysteine residue, is important for the sing p21 GDS action.

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Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

Cited by 46 publications

References 30 publications

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

Examination of the Coordinate Effects ofPseudomonas aeruginosaExoS on Rac1

A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21.

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