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J B Gibbs

Publications and source records attributed to J B Gibbs.

At least 91 records · Page 5Linked to original sources
PubMed1989

Xenopus oocyte germinal-vesicle breakdown induced by [Val12]Ras is inhibited by a cytosol-localized Ras mutant.

The GTPase-activating protein (GAP) has been postulated to function either as a negative regulator or as a possible target protein of Ras in mammalian cells and Xenopus oocytes. Ras must be localized in the plasma membrane of vertebrate cells to function, but GAP is localized in the cytosol. To test whether Ras function depends on a cytosolic factor such as GAP, we microinjected into Xenopus oocytes a form of Saccharomyces cerevisiae RAS1 ([Leu68]RAS1 terminated at residue 185, called [Leu68]RAS1(term.] that lacks the consensus membrane localization site, does not respond to GAP in a GTPase assay, but binds to GAP 100-fold more tightly than [Val12]Ras. [Leu68]RAS1(term.) alone did not stimulate oocyte germinal-vesicle breakdown. Instead, [Leu68]RAS1(term.) was observed to inhibit the action of insulin-like growth factor 1 or microinjected [Val12]Ras but not the action of progesterone as monitored by germinal-vesicle breakdown. Coinjection of purified mammalian GAP with [Leu68]RAS1(term.) reduced the inhibition of [Val12]Ras-stimulated germinal-vesicle breakdown. The results raise the possibility that a cytosolic factor is required for the action of [Val12]Ras in Xenopus oocytes and that this factor is either GAP or another protein with which GAP can compete for binding to [Leu68]RAS1(term.).

Animals↗
PubMed1989

The ras oncogene--an important regulatory element in lower eucaryotic organisms.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.

Adenylyl Cyclases↗
PubMed1988

Cloning of bovine GAP and its interaction with oncogenic ras p21.

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

Amino Acid Sequence↗
PubMed1988

Inhibition of yeast adenylate cyclase by antibodies to ras p21.

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.

Adenylyl Cyclases↗
PubMed1988

Purification of ras GTPase activating protein from bovine brain.

In cytosolic extracts of bovine brain, we detected ras GTPase activating protein (GAP) activity that stimulated the GTP hydrolytic activity of normal c-Ha-ras p21 but not that of the oncogenic [Val12]p21 variant. GAP was purified 19,500-fold by a five-column procedure involving DEAE-Sephacel, Sepharose 6B, orange dye and green dye matrices, and Mono Q resins. A single major protein band of 125 kDa was observed on NaDodSO4/polyacrylamide gels that correlated with the elution of GAP activity on Mono Q. Purified GAP was devoid of inherent GTP hydrolytic activity, suggesting that it was a regulator of ras intrinsic GTPase activity. Under submaximal velocity conditions, the second-order rate constant of GTP hydrolysis at 24 degrees C for p21-GTP + GAP (4.5 X 10(6) M-1.sec-1) was at least 1000-fold greater than that for [Val12]p21-GTP + GAP (less than 3 X 10(3) M-1.sec-1).

Animals↗
PubMed1988

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.

Adenylyl Cyclases↗
PubMed1987

Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells.

We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in [32P]orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells. Whereas S. cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions. In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97. Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity. Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.

Animals↗
PubMed1987

CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

Adenylyl Cyclases↗
PubMed1987

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

Adenylyl Cyclases↗
PubMed1987

Molecular approaches towards an anti-ras drug.

The ras proteins have intrinsic biochemical properties that are similar to those of the guanine nucleotide binding regulatory proteins (G-proteins). Increased oncogenic potential results from amino acid substitutions that, by altering either the intrinsic GTPase activity or the GDP/GTP exchange rate, would lead to an increase in the level of the ras-GTP complex. The functional similarity between the ras oncogene proteins and the G-proteins suggests several mechanisms for an anti-ras drug. Anti-ras drugs could act either by retarding the formation of the biologically active GTP complex of the protein or by preventing the ras protein from interacting with its yet-to-be-identified target. Mutagenesis studies of Harvey (Ha) ras have identified the residues involved in GDP and GTP binding, the residues that constitute the epitope for the neutralizing antibody Y13-259 (63-73) and a region (32-40) that is required for effector action. Computer modeling combined with immunological characterization has suggested some structural properties of this putative 'effector' region.

Antineoplastic Agents↗
PubMed1986

Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21.

To identify the amino acid residues of the Harvey (Ha) ras-encoded protein that are involved in protein-protein interactions, we have created a series of mutant Ha-ras proteins. In particular, amino acid substitutions have been introduced within two regions, residues 32-42 and 61-80, that are conserved among ras proteins from different species. We observed that amino acid substitutions at positions 35, 36, 38, 40, and, to a lesser extent, 39 and 78 reduce the biological potency of Ha-ras protein in both mammalian and Saccharomyces cerevisiae cells, without noticeably affecting the known intrinsic biochemistry of these proteins. The reduction of in vivo activity for these mutant ras proteins correlates with their reduced ability to stimulate yeast adenylate cyclase. The ras-protein-neutralizing antibody Y13-259 binds to six residues: Glu-63, Ser-65, Ala-66, Met-67, Gln-70, and Arg-73. Single substitutions for these residues reduce Y13-259 antibody binding by at least a factor of 1000 but do not significantly affect biological activity. These data are discussed in terms of the model for Ha-ras protein based on the structure of the elongation factor EF-Tu-GDP complex.

Adenylyl Cyclases↗
PubMed1986

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.

Animals↗
PubMed1986

Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.

We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.

Animals↗
PubMed1986

Suppressors of the ras2 mutation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.

Adenylyl Cyclases↗
PubMed1984

Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells.

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6-8 h after an initial 2-3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.

Animals↗
PubMed1984

Calcium dependence for increased antizyme inhibitory activity of ornithine decarboxylase in rat glioma cells.

Ornithine decarboxylase activity was inhibited by the antizyme inhibitor protein in extracts from C6-2B rat glioma cells. Antizyme activity in C6-2B cells was increased 3- to 10-fold by micromolar concentrations of putrescine, spermidine and spermine. The calcium chelator EGTA (pCa 6.4) inhibited basal and polyamine-stimulated antizyme activity, and this inhibition was prevented by concurrent incubation with calcium, but not with magnesium. EGTA appeared to block antizyme synthesis, because the half-life values of antizyme activity in the presence of EGTA or cycloheximide were similar (121-143 min). Also, calcium readdition rapidly reversed EGTA inhibition of antizyme activity by a mechanism which could be blocked by cycloheximide. The ability of EGTA to inhibit spermidine-stimulated antizyme activity was not due to reduced spermidine uptake, because EGTA actually stimulated [3H]spermidine accumulation in the trichloroacetic acid-soluble fraction of C6-2B cells after 3 h.

Animals↗