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At least 19 recordsLinked to original sources
PubMed1992

ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.

ADP Ribose Transferases↗
PubMed1992

Intracellular localization of the P21rho proteins.

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

Amino Acid Sequence↗
PubMed1992

Post-translational modifications of p21rho proteins.

Post-translational modifications of the ras proteins, which are required for plasma membrane localization and biological function of the proteins, have been shown to include prenylation and carboxymethylation at the carboxyl terminal cysteine residue of the cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) box. In addition, p21Ha-ras and p21N-ras, but not p21K-ras (B), are palmitoylated. The three mammalian rho proteins (A, B, and C) are also members of the ras superfamily but have distinct biological activities and different intracellular distributions from p21ras. Analysis showed all three rho proteins are modified by a COOH-terminal carboxymethylation similar to p21ras, whereas p21rhoC labeled with [3H]mevalonic acid in vivo revealed the presence of a C20 prenoid, similar to that already described for p21rhoA. However, in vivo and in vitro studies of p21rhoB showed this protein to be modified by both C15 and C20 prenoids. Mutation of C193 in the CAAX box abolished prenylation, whereas mutation of the adjacent C192 resulted in a significant reduction in the amount of the C20, but not C15 prenoid, recovered from p21rhoB. In vivo labeling studies with [3H]palmitic acid and mutational analysis showed that both cysteine residues at 189 and 192 upstream of the CAAX box in p21rhoB are sites for palmitoylation. We conclude that there are different populations of post-translationally modified p21rhoB in the cell and that the sequence specificity for geranylgeranyl- and farnesyltransferases may be more complicated than previously proposed.

Amino Acid Sequence↗
PubMed2026

11-O-galloylbergenin alleviates LPS-stimulated inflammation in RAW 264.7 macrophages by targeting Grb2, RhoA, and Cdc42 in the RAS signaling pathway.

OBJECTIVE: This study aimed to explore the anti-inflammatory mechanism of 11-O-galloylbergenin in macrophages. METHODS: Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were treated with 11-O-galloylbergenin. Cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cytokine secretion was measured by enzyme-linked immunosorbent (ELISA) assay. Data-independent acquisition (DIA)-based proteomics, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, western blotting, and molecular docking were also performed. RESULTS: 11-O-Galloylbergenin (≤50 μg/mL) was non-cytotoxic and significantly suppressed tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) secretion. Proteomics analysis identified 209 differentially expressed proteins (DEPs) that showed opposite expression trends before and after 11-O-galloylbergenin treatment. Seven of these DEPs were enriched in the RAS pathway: RhoA, Cdc42, Grb2, RalB, Calm3, Gnb2, and Pla2g4a. Western blotting confirmed that 11-O-galloylbergenin downregulated RhoA, Cdc42, and Grb2 expression. Molecular docking revealed good binding affinity of 11-O-galloylbergenin to RhoA, Cdc42, and Grb2. CONCLUSION: 11-O-Galloylbergenin alleviates LPS-stimulated inflammation in RAW 264.7 macrophages by inhibiting the RAS signaling pathway.

Animals↗
PubMed1992

ADP-ribosylation by Clostridium botulinum C3 exoenzyme increases steady-state GTPase activities of recombinant rhoA and rhoB proteins.

ADP-ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady-state GTP hydrolysis by 50 to 80%. ADP-ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti-C3 antibody or heat inactivation of C3. In contrast, GTP hydrolysis by Ile-41 rhoA or Ha-ras, which are no substrates for the transferase, were not affected by C3. ADP-ribosylation facilitated the [3H]GDP release and subsequently, the binding of [3H]GTP to rhoA. The data indicate that the increase in the steady-state GTPase activity by ADP-ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP-binding proteins.

ADP Ribose Transferases↗
PubMed1992

A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.

ADP Ribose Transferases↗
PubMed1992

A novel type of regulatory protein for the GDP/GTP exchange reaction of rho p21, a ras p21-like small GTP-binding protein, in rabbit intestine.

A novel regulatory protein for rho p21, a ras p21-like GTP-binding protein (G protein), was purified from the cytosol fraction of rabbit intestine. This protein, designated as rho GDP dissociation inhibitor (GDI), regulated the GDP/GTP exchange reaction of rho p21 by inhibiting the dissociation of GDP from and subsequent binding of GTP to it. rho GDI did not affect the GTPase activity of rho p21. rho GDI formed a complex with the GDP-bound form of rho p21 but not the GTP-bound form. rho GDI was inactive for other small G proteins including ras p21, smg p21 and smg p25A. These results indicate that rho GDI is a novel type of regulatory protein for the GDP/GTP exchange reaction of rho p21.

Animals↗
PubMed1992

A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation.

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.

ADP Ribose Transferases↗
PubMed1992

Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle.

The GDP/GTP exchange reaction of rho p21, a member of ras p21-related small GTP-binding protein superfamily, is regulated by two stimulatory GDP/GTP exchange proteins (GEPs), named smg GDS and rho GDS, and by one inhibitory GEP, named rho GDI. In bovine aortic smooth muscle, rho GDS and rho GDI were major GEPs for rho p21, and the rho GDI activity on the GDP/GTP exchange reaction of rho p21 was stronger than the rho GDS activity in their simultaneous presence. Moreover, in the crude cytosol, the GDP-bound form of rho p21 was complexed with rho GDI but not with rho GDS. These results, together with our recent finding that rho p21 is involved in the vasoconstrictor-induced Ca2+ sensitization of smooth muscle contraction, suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the stimulatory action of rho GDS, eventually leading to the rho p21 activation, in the signaling pathways of the vasoconstrictor receptors in smooth muscle.

Animals↗
PubMed2003

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity.

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

3T3 Cells↗
PubMed1992

Function of the post-translationally modified C-terminal region of rho p21.

rhoA p21, a ras p21-like small GTP-binding protein, purified from bovine aortic smooth muscle is similarly modified at its C-terminal region as described for ras p21s. In this study, I examined the role of the post-translational modifications of the C-terminal region of rhoA p21 by comparing bovine rhoA p21 with bacterially-produced rhoA p21 because the bacterial protein was not modified. Bovine rhoA p21 bound to plasma membranes, but bacterial rhoA p21 did not. Both the stimulatory and inhibitory GDP/GTP exchange proteins for bovine rhoA p21 were inactive for bacterial rhoA p21. On the other hand, the GTPase activating protein for bovine rhoA p21 was also active for bacterial rhoA p21. These results indicate that the post-translational modifications of the C-terminal region of bovine rhoA p21, which are absent in bacterial rhoA p21, are essential for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GTPase activating protein.

Animals↗
PubMed1992

Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.

In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction. GTP gamma S alone did not induce the contraction in the absence of Ca2+. This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily. The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN. smg p21B, another small GTP-binding protein, was inactive. EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle. The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction. smg p21B was inactive. These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.

Adenosine Diphosphate Ribose↗
PubMed1992

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.

Animals↗
PubMed1992

Molecular cloning of the human cDNA for a stimulatory GDP/GTP exchange protein for c-Ki-ras p21 and smg p21.

We have previously purified smg GDP dissociation stimulator (GDS) from bovine brain and isolated its cDNA from a bovine brain cDNA library. smg GDS stimulates the GDP/GTP exchange reaction of a group of small GTP-binding proteins (G proteins), including at least c-Ki-ras p21, smg p21A, smg p21B, rhoA p21 and rhoB p21, by stimulating the dissociation of GDP from and the subsequent binding of GTP to each small G protein. In this study, we have isolated and sequenced the cDNA of smg GDS from a human brain cDNA library using the cloned bovine smg GDS cDNA. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,122. Human smg GDS shares 93% nucleotide and 96% amino acid sequence homologies with bovine smg GDS. The isolated cDNA is expressed in Escherichia coli, and the encoded protein shows the physical and functional properties similar to those of bovine smg GDS.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum.

We purified a novel ADP-ribosyltransferase produced by a Clostridium limosum strain isolated from a lung abscess and compared the exoenzyme with Clostridium botulinum ADP-ribosyltransferase C3. The C. limosum exoenzyme has a molecular weight of about 25,000 and a pI of 10.3. The specific activity of the ADP-ribosyltransferase is 3.1 nmol/mg/min with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the tryptic peptides revealed about 70% homology with C3. The novel exoenzyme modifies selectively the small GTP-binding proteins of the rho family in human platelet membranes presumably at the same amino acid (asparagine 41) as known for C3. Recombinant rhoA and rhoB serve as substrates for C3 and the C. limosum exoenzyme. Whereas recombinant rac1 protein is only marginally ADP-ribosylated by C3 or by the C. limosum exoenzyme in the absence of detergent, in the presence of 0.01% sodium dodecyl sulfate rac1 is modified by C3 but not by the C. limosum exoenzyme. Recombinant CDC42Hs protein is a poor substrate for C. limosum exoenzyme and is even less modified by C3. The C. limosum exoenzyme is auto-ADP-ribosylated in the presence of 0.01% sodium dodecyl sulfate by forming an ADP-ribose protein bond highly stable toward hydroxylamine. The data indicate that ADP-ribosylation of small GTP-binding proteins of the rho family is not unique to C. botulinum C3 ADP-ribosyltransferase but is also catalyzed by a C3-related exoenzyme from C. limosum.

ADP Ribose Transferases↗
PubMed1992

Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties.

Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations. Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol.min-1.mg protein-1 (turnover number approximately 0.39 min-1 at 37 degrees C), which is 40-fold higher than H-ras. The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min-1 for rac1 vs 0.007 min-1 for H-ras (at 20 degrees C). Rac1 also released bound GDP faster than H-ras (koff.GDP = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20 degrees C). In contrast, rac1 released bound guanosine 5'-[gamma-thio]triphosphate (GTP[S]) at a slower rate than H-ras (koff.GTP[S] approximately 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20 degrees C). Rac1 was a very good substrate for in vitro geranylgeranylation (C20) but not for farnesylation (C15), whereas the converse is true for H-ras. Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine. This protein (referred to as [Thr115----Asn]rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras. The koff.GDP of [Thr115----Asn]rac1 was reduced to that of H-ras, but [Thr115----Asn]rac1 exhibited essentially identical kcat (0.13 min-1 at 20 degrees C) and koff-GTP[S] (0.03 min-1 at 20 degrees C) values as the wild-type protein. Thus, the region(s) in rac1 which control the dissociation of GTP[S] (and presumably GTP) do not entirely coincide with those controlling GDP dissociation. Biochemical analysis of [Thr115----Asn]rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.

Adenosine Diphosphate Ribose↗
PubMed1992

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.

Amino Acid Sequence↗