Human H-ras Protein Research Articles

Arglabin-DMA, a plant derived sesquiterpene, inhibits farnesyltransferase.

Arglabin [1(R),10(S)-epoxy-5(S),5(S),7(S)-guaia-3(4),11(13)-dien-6, 12-olide], a sesquiterpene gamma-lactone is isolated from Artemisia glabella, a species of wormwood endemic to the Karaganda region of Kazakstan. The compound has been modified to render it water-soluble through addition of a dimethylaminohydrochloride group to the C(13) carbohydride moiety to yield Arglabin-DMA. Arglabin-DMA is a registered antitumor substance in the Republic of Kazakstan. Previously, we have shown that this compound prevents protein farnesylation without altering geranylgeranylation. We now report that Arglabin-DMA inhibits the incorporation of [(3)H]farnesylpyrophosphate into human H-ras protein by FTase with an IC(50) of no greater than 25 microM. Kinetic studies show that the phosphorylated form of this compound competitively inhibits the binding of farnesyl diphosphate to FTase. This mechanism of action is different from other reported peptidomimetic FTIs which lower the affinity of ras protein to FTase. Our in vitro studies confirm that Arglabin-DMA inhibits post-translational modification of ras protein in cells. Arglabin-DMA inhibits anchorage-dependent proliferation of NB cells (IC50=10 microg/ml) and inhibits anchorage-independent growth of NB and KNRK cells with about the same IC(50). Soft-agar colony formation assay of H-ras and K-ras transformed cells show IC(50)s to be 2 and 5 microg/ml, respectively. In primary cultures of human tumor cells, Arglabin-DMA inhibits cell proliferation of a variety of tumor types with IC(90)s in the range of 0.85 to 5.0 microg/ml. Because of these pharmacologic properties, we propose that Arglabin-DMA is suitable for the treatment of ras related malignancies.

Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p.

Small GTPases of the Ypt/Rab family are regulators of vesicular protein trafficking in exo-and endocytosis. GTPase-activating proteins (GAP) play an important role as down regulators of GTPases. We here report the molecular cloning of a novel GAP-encoding gene (GYP7, for GAP for Ypt7) by high expression from a Saccharomyces cerevisiae genomic library. The GYP7 gene encodes a hydrophilic protein with a molecular mass of 87 kDa. Comparison of its primary sequence with that of the three other known GAPs for transport GTPases, the yeast Gyp6 and Gyp1 proteins and the Rab3A-GAP from rat brain, shows similarity between the yeast GAPs only. Like GYP6 and GYP1, GYP7 is not essential for yeast cell viability. Gyp7p was able to most effectively accelerate the intrinsic GTPase activity of Ypt7p. It was also active, but to a lesser extent, on Ypt31p, Ypt32p and Ypt1p. Ypt6p, Sec4p and the human H-Ras protein did not serve as substrates. We also report the identification and cloning of a gene from the dimorphic yeast Yarrowia lipolytica that encodes a protein whose primary structure and biochemical activity are significantly related to those of Gyp7p from baker's yeast.

Mechanism of activation of the Caenorhabditis elegans ras homologue let-60 by a novel, temperature-sensitive, gain-of-function mutation.

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25 degrees, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15 degrees, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60 (L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.

A Human Protein Selected for Interference with Ras Function Interacts Directly with Ras and Competes with Raf1

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins.

Ras proteins appear to have two distinct downstream effectors, adenylyl cyclase in Saccharomyces cerevisiae and a product of raf-1 protooncogene in higher organisms. We found that in vitro activation of adenylyl cyclase by yeast Ras2 and human H-Ras proteins is subject to competitive inhibition by its leucine-rich repeats domain and by the N-terminal regulatory domain of human Raf-1 protein. Kinetic analyses of the inhibition patterns enabled us to determine exact dissociation constants (Kd) of the two polypeptides for Ras2 and H-Ras. The leucine-rich repeats domain bound to the posttranslationally modified Ras2 with the Kd of approximately 13 nM, which was close to the value (7 nM) of the whole adenylyl cyclase. The Kd of Raf-1 for the modified H-Ras, 3.5 nM, was significantly lower than that for Ras2, 24 nM, whereas adenylyl cyclase bound preferentially to Ras2. Similar inhibition was also observed in vivo by suppression of RAS2Val-19-dependent heat shock sensitivity and of Ras-dependent cAMP response to glucose upon overexpression of Raf-1 in yeast. These results indicate that the leucine-rich repeats domain contains the Ras protein-binding site and that Raf-1 and adenylyl cyclase, sharing no structural homology with each other, bind to a similar, if not identical, region of Ras with comparable affinities.

Mass spectrometric detection of the noncovalent GDP-bound conformational state of the human H-ras protein

Mass spectrometric detection of the noncovalent GDP-bound conformational state of the human H-ras protein

Molecular model of the G protein alpha subunit based on the crystal structure of the HRAS protein.

A structural model of guanine nucleotide-binding regulatory protein alpha subunits (G alpha subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21ras). Because of low overall sequence similarity, structural and functional constraints were used to align the G alpha consensus sequence with that of p21ras. The resulting G alpha model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the beta gamma subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G alpha model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21ras, the model assigns specific residues in G alpha required for binding the guanosine (G-box) and phosphates (PO4-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G alpha model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.

Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins.

Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.

The oncogenic activation of human p21ras by a novel mechanism.

Single amino acid changes were introduced into normal (non-oncogenic) and activated forms of the human H-ras protein at a position (residue 116) proposed on structural grounds to represent a contact site with guanine nucleotides. Substitutions at this site could significantly reduce the ability of both forms to bind and hydrolyze guanosine 5'-triphosphate; these substitutions, however, did not necessarily diminish the transforming capacity of activated derivatives. One substitution that severely impairs these functions activated the transforming potential of the otherwise normal polypeptide.

RAS proteins can induce meiosis in xenopus oocytes

Injection of human H-ras protein induces maturation of Xenopus oocytes; that is, progression from prophase to metaphase of meiosis. The oncogenic protein encoded by H-rasval12 is nearly a 100-fold more potent than the protein encoded by the wild-type gene. We do not observe any measurable increase or decrease in cyclic AMP concentration in injected oocytes, and the effects of H-ras protein are only partially blocked by cholera toxin. Our results suggest that not all, if any, of the effects of H-rasval12 protein in this system are mediated by adenylate cyclase.

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.