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Query: UNIPROT:P06889 (Mol)
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Transformation of cloned rat embryo fibroblast (CREF) cells with the wild-type 5 adenovirus (wtAd5) transforming genes E1A and E1B (which extend from 0 to 11.2 map units) results in morphologically transformed cells that exhibit an increased saturation density in monolayer culture and display an anchorage-independent phenotype. WtAd5-transformed CREF (wtAd5 CREF) cells do not, however, induce tumors when injected subcutaneously into athymic nude mice or syngeneic Fischer rats. We have analyzed the effect of the ras oncogene and site-specific mutants in the ras oncogene that result in p21 proteins with altered biochemical properties on the oncogenic and metastatic properties of singly (ras) and doubly (ras + wtAd5) transformed CREF cells. Transformants expressing the wild-type ras p21 protein and ras mutants producing p21 proteins that retained GTP-binding properties grew in agar, induced tumors in nude mice and syngeneic rats, and metastasized to the lungs of rats when injected into their tail veins. In contrast, cells transformed with the ras mutant 116K (which contains a mutation at residue 116 that produces a Lys instead of an Asn and does not bind GTP or induce CREF cells to grow in agar) did not become morphologically transformed and were not oncogenic when injected subcutaneously into either nude mice or Fischer rats; further, such cells were not metastatic when injected into the tail veins of Fischer rats. When the wild-type ras or the ras mutants, including 116K, were expressed in nontumorigenic E1A-plus-E1B-expressing wtAd5 CREF cells, transformed cells induced tumors in both types of animals. The CREF cells doubly transformed with 116K + wtAd5, unlike transformants containing the wild-type ras and the other ras mutants that still retained GTP binding, were still unable to induce lung metastases. In addition, 116K + wtAd5-transformed CREF cells also did not display any alterations in morphology distinguishable from wtAd5 CREF cells and were not able to grow in agar with increased efficiency. These results indicate that the loss of GTP-binding ability by this mutant p21 ras protein eliminated the ability of these proteins to induce an oncogenic phenotype in an immortal but normal CREF cell line. However, the mutant ras could cooperate with wtAd5 transforming genes in transformed CREF cells to make these cells progress to an oncogenic (but not metastatic) phenotype.
Mol Carcinog 1992
PMID:Induction and progression of the transformed phenotype in cloned rat embryo fibroblast cells: studies employing type 5 adenovirus and wild-type and mutant Ha-ras oncogenes. 155 10

E. coli ribosomes with alterations in S12 leading to streptomycin resistance (SmR), dependence (SmD) and pseudodependence (SmP) were studied with the quench-flow technique. Kinetic changes at the various steps of the elongation cycle were identified. The rate of hydrolysis of GTP in the ternary complex in the ribosomal A-site is decreased drastically in SmD and moderately in SmP in relation to wild-type ribosomes. Addition of streptomycin restores much of the wild-type behaviour. The SmD, SmP and SmR ribosomes have an enhanced GTP-hydrolysis idling reaction on EF-Tu, which is correlated with how aggressive proofreaders these ribosomes are in steady-state assays. We use our in vitro findings to discuss the in vivo physiology of these mutants as well as mechanistic features of E. coli translation.
J Mol Biol 1992 Apr 20
PMID:Kinetic properties of Escherichia coli ribosomes with altered forms of S12. 156 65

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
Mol Cell Biol 1992 May
PMID:Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. 156 38

Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2. We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo. Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins. The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity. On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2. Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.
Mol Cell Biol 1992 May
PMID:The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo. 156 42

Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTP gamma [35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated Gi protein. An increase in GTP hydrolysis (approximately 50% over basal activity) and GTP gamma [35S] binding (approximately 25-60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP-ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTP gamma [35S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.
Mol Reprod Dev 1992 Apr
PMID:Activation of a G protein in mouse sperm by the zona pellucida, an egg-associated extracellular matrix. 157 Nov 63

Chronic inflammation of the colon and the rectum was induced by intracolonic administration of 25 mg trinitrobenzoic sulfonic acid (TNB) in 0.25 ml 30% ethanol. Three weeks after TNB administration the colon and the rectum showed transmural, granulomatous inflammation which had many similarities to Crohn's disease and furthermore to the morphological and functional changes which occur in early phases of postischemic intestinal damage. In the colon of TNB-treated animals the ATP and GTP levels were markedly decreased. The accumulation of thiobarbituric acid-reactive substances (TBA-RS) demonstrated a free radical-mediated component of the tissue damage. Treatment with oxypurinol radical scavenger and xanthine oxidoreductase inhibitor diminished the morphological changes, the loss of energy-rich nucleotides and the TBA-RS accumulation.
Cell Mol Biol 1992 Apr
PMID:Protective influence of oxypurinol on the trinitrobenzene sulfonic acid(TNB) model of inflammatory bowel disease in rats. 157 48

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
Mol Biol Cell 1992 May
PMID:Ras-related GTPases and the cytoskeleton. 161 Nov 53

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase. The inhibition was dependent on the receptor density expressed, increasing from 60% at low density (45 fmol/mg) to 90% at a density higher than 280 fmol/mg. The pharmacology of the 5-HT1A receptor was studied, with particular attention being paid to the behavior of some agonists. These pharmacological characteristics are similar to those of 5-HT1A receptors in hippocampus but different from those of 5-HT1A in cerebral cortex. Analysis of the potencies and efficacies of the full agonist 5-HT and the partial agonist ipsapirone, as a function of receptor density in the three cellular populations used, revealed that 1) the efficacies of the full and partial agonists increased with the receptor density; 2) the EC50 values of the full and partial agonists were not shifted to the left when the receptor density was increased (based on the increase in efficacy and considering the classical pharmacological models of receptor-drug action, a 9-10-fold shift was expected); and 3) the ratio between the efficacies of the full agonist 5-HT and the partial agonist ipsapirone was not modified when the receptor concentration was increased or when the GTP-binding protein availability was decreased. The results indicate that neither the classical nor the operational model of drug-receptor action can be used to describe the coupling of 5-HT1A receptors to adenylyl cyclase in transfected NIH-3T3 cells. One of the explanations could be that 5-HT1A receptors and GTP-binding proteins are coupled in functional domains (almost precoupled), rather than distributed in homogeneous compartments in which they are free to diffuse.
Mol Pharmacol 1992 Jun
PMID:Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase. 161 16

We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.
Mol Cell Biol 1992 Jul
PMID:Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae. 162 Jan 8

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.
Mol Cell Biol 1992 Jul
PMID:Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. 162 Jan 32


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