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At least 19 recordsLinked to original sources
PubMed1994

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.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins.

Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.

Binding Sites↗
PubMed1992

Prenoids and palmitate: lipids that control the biological activity of Ras proteins.

Ras proteins can be modified by two types of lipids--an isoprenoid and the fatty acid palmitate. These lipids help the otherwise cytoplasmic Ras protein to interact with the plasma membrane of a cell. The biological consequences of this association between Ras and membranes are dramatic, and can alter a cell's behavior from normal growth into malignancy. The scope and limits of our knowledge of the steps, structures and enzymes involved in this molecular transformation from soluble inactivity to membrane-bound potency are offered below. The prospects of regulating Ras function by controlling its intracellular location provides a tantalizing opportunity to translate research into a novel therapeutic reality.

Amino Acid Sequence↗
PubMed2002

Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization of yeast Ras proteins.

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, and X can be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes, ERF2 and ERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

Acyltransferases↗
PubMed1990

Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast.

Alkyl and Aryl Transferases↗
PubMed2006

Activation of Ras proteins by Ras guanine nucleotide releasing protein family members.

Ras guanine nucleotide releasing proteins (RasGRPs) function as guanine nucleotide exchange factors for Ras proteins. Thus, RasGRPs are direct activators of Ras proteins and contribute an important role in various cell-signaling pathways that are regulated by the activation state of Ras proteins. RasGRPs are regulated by the second messengers diacylglycerol and intracellular calcium and are also known as CalDAG-GEFs or calcium and diacylglycerol-regulated guanine nucleotide exchange factors. RasGRPs couple signaling events that generate these second messengers in the cell into activation of signaling pathways that are regulated by Ras. RasGRPs, therefore, increase the repertoire of extracellular stimuli that lead to activation of Ras. Analyzing the regulation of RasGRP activity should continue to play an important role in understanding the mechanisms by which signal transduction pathways use RasGRP proteins to activate Ras proteins in cells.

Animals↗
PubMed1986

Mammalian G proteins: models for ras proteins in transmembrane signalling?

How do the p21v-ras proteins and their normal cellular counterparts regulate cell function? What is the molecular basis of action of these proteins? Biochemical, structural and functional similarities between the ras proteins and the vertebrate G proteins offer clues that may help to answer such questions. The G proteins couple a wide array of extracellular signals to regulation of a number of enzyme effectors, including adenylate cyclase, retinal cGMP phosphodiesterase and phospholipase-C. The RAS1 and RAS2 proteins of yeast regulate adenylate cyclase, whereas their close mammalian homologues, the p21ras proteins, do not. Both the ras and the G proteins are located at the cytoplasmic face of the plasma membrane and bind and hydrolyse GTP. Patchy amino acid sequence homologies between the two groups of proteins suggest a common evolutionary origin and common structural features, particularly in the GTP binding domain. In the GTP bound state both proteins are 'on' or activated, and each exhibits an intrinsic GTPase activity that turns off the active state. The analogies between the G and ras proteins suggest that the latter may also couple signal detector and enzymatic effector elements, and suggest strategies for identifying them.

Fungal Proteins↗
PubMed1990

Common modifications of trimeric G proteins and ras protein: involvement of polyisoprenylation.

The heterotrimeric guanine nucleotide-binding regulatory proteins act at the inner surface of the plasma membrane to relay information from cell surface receptors to effectors inside the cell. These G proteins are not integral membrane proteins, yet are membrane associated. The processing and function of the gamma subunit of the yeast G protein involved in mating-pheromone signal transduction was found to be affected by the same mutations that block ras processing. The nature of these mutations implied that the gamma subunit was polyisoprenylated and that this modification was necessary for membrane association and biological activity. A microbial screen was developed for pharmacological agents that inhibit polyisoprenylation and that have potential application in cancer therapy.

Amino Acid Sequence↗
PubMed1988

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants.

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).

Base Sequence↗
PubMed1994

Haemoglobin inhibits GTP-hydrolysis and GDP/GTP-exchange activities of a low M(r) GTP-binding protein, ras p21.

Haemoglobin was observed to inhibit the GDP/GTP-exchange activity of ras protein (ras p21) by measurement of [3H]GDP-dissociation activity in time- and dose-dependent manners. Haemoglobin also inhibited the [32P]GTP-hydrolysis activity of ras p21 time- and dose-dependently. These inhibitory effects of haemoglobin were lost after incubation of haemoglobin at 80 degrees C for 3 min. Globin showed limited inhibition on the [32P]GTP-hydrolysis activity of ras p21, and haemin had no effect, indicating that the ternary tetrameric structure of haemoglobin is essential for the inhibitory effects on ras p21 activities. Methaemoglobin also inhibited both [3H]GDP-dissociation and [32P]GTP-hydrolysis activities of ras p21 in a very similar manner to that by haemoglobin. The obtained results strongly suggest that haemoglobin suppresses the physiological function(s) of ras p21 in vivo inhibiting both [32P]GTP-hydrolysis and GDP/GTP-dissociation of ras p21 in erythrocytes.

Dose-Response Relationship, Drug↗
PubMed1987

Regulation of membrane turnover by ras proteins.

Because ras oncogenes mediate abnormal cellular growth, ras proteins have been presumed to play a role primarily in growth control. The biological function of ras proteins may, however, prove to be much more diverse: ras proteins may be involved in cellular functions that control endocytosis and/or exocytosis.

Cell Membrane↗
PubMed1988

Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins.

The ras proteins represent a unique example of membrane proteins which apparently do not utilize the secretory pathway for their membrane localization. Instead, it is believed that palmitic acid, covalently attached to the protein, acts as an anchor to the membranes. Recent identification of yeast mutants defective in the processing of the ras proteins has provided a novel approach for defining these biosynthetic processes. We report here the characterization of yeast DPR1, a gene essential for the processing of the ras proteins. The sequence of the gene indicates that it encodes a protein of 431 amino acids which contains no significant homology with any known proteins. It is a relatively hydrophilic protein with no apparent hydrophobic stretches. The C-terminal half of the encoded protein has an unusually high content of cysteine. The DPR1 gene product has been identified in a cell-free translation system as a protein having an apparent molecular weight of 43 kd. This represents the first step in the investigation of a novel protein-processing pathway, one that is distinct from the secretory pathway.

Amino Acid Sequence↗
PubMed1989

Structural and functional properties of ras proteins.

The ras proteins belong to a family of related polypeptides that are present in all eukaryotic organisms from yeast to human. Their extraordinary evolutionary conservation suggests that they have essential cellular functions, although their exact role remains unknown. Mutations in specific amino acids and overexpression of normal proteins have been linked to altered proliferation and/or differentiation and, particularly, to neoplastic processes. Mature ras proteins are located on the inner side of the plasma membrane, and their biochemical properties include binding and exchange of guanine nucleotides and GTPase activity. The favored hypothesis for ras function is that these proteins exist in an equilibrium between an inactive conformation (p21.GDP) and an active conformation (p21. GTP) in which they are able to interact with their as yet unknown cellular target or targets. Similarities in cellular location, structure, and biochemistry with other known regulatory (G) proteins suggest that they play a role in transduction of signals from the cell surface. The elucidation of the crystal structure of normal and transforming ras proteins and the identification of cellular proteins that interact directly with them (GAP, CDC25) or suppress some of their biological effects (Krev-1) have opened new avenues in the search for their elusive cellular targets and in the elucidation of the functional role of ras gene products.

Amino Acid Sequence↗
PubMed1984

Developmental regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras protein.

We have cloned, sequenced, and examined the regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras proteins. The Dictyostelium, yeast, and mammalian proteins have homologous N-terminal regions and less conserved C-terminal regions. We have used DNA probes and a polyclonal antibody to examine the differential accumulation of ras RNA and protein through development. The gene encodes two mRNAs (0.9 and 1.2 kb) that are differentially expressed. The 1.2 kb RNA is found in vegetative cells and disappears rapidly upon initiation of development. Later, both RNAs accumulate preferentially in prestalk cells. The level of the Dd-ras protein remains constant until early culmination and then decreases. Like other prestalk genes, Dd-ras can be induced with cAMP in the absence of cell contact. When aggregated cells are dissociated, both mRNAs decrease. Upon addition of cAMP, the 1.2 kb mRNA reaccumulates at a higher level than that in normal developing cells. The presence of the Dd-ras protein in vegetative cells corroborates other reports suggesting a possible function during cell growth. The sustained level of Dd-ras protein in prestalk cells suggests an additional role during differentiation.

Amino Acid Sequence↗
PubMed1991

ras proteins and the ras-related signal transduction pathway.

Mammalian ras genes may naturally acquire oncogenic transformation potential through some point mutations which result in the impairment of the normal ras protein functions, and which are localised in codons 12, 13 or 61. Mutationally activated ras alleles were found in a wide variety of human and carcinogen (including radiation)-induced animal malignancies. In man, myeloid leukemias are often associated with the presence of a mutationally activated ras gene (for review, see Bos JL (1989), Cancer Res 49:4682-4689). However, we failed till now in our attempts to detect oncogenic ras mutations in radiation-induced mouse myeloid leukemias. We thus have the feeling that ras might perhaps participate to tumorigenesis through another mechanism provoking a deregulation of the ras protein functions. In order to help evaluate such a possibility, we give here a very concise overview of the properties of the ras proteins and of their regulation by a variety of still hypothetical molecular switches. This overview does not include bibliographic references. Indeed, we gathered much of the information described below at the Cold Spring Harbor Symposium on "Function and Evolution of ras Proteins", May 9-13, 1990. Communications presented at Cold Spring Harbor Symposia may contain preliminary data and should not be cited in bibliographies. Another voluntary omission in this overview is that, for the sake of simplicity, we do not mention whether the data were obtained from experiments performed on H-, K- or N-ras. Details can be found in the published book of abstracts.

Animals↗
PubMed1997

[Isoprenylated proteins and cell proliferation: regulators and effectors of Ras proteins].

Ras proteins play a central role in the control of cellular proliferation. They are 189 amino acid monomeric GTP-binding proteins that cycle between an inactive GDP-bound and the active GTP-bound state, and carry a slow intrinsic GTPase activity. Ras proteins are activated by growth promoting signals incoming from receptor tyrosine kinases via SH2 domain and SH3 domain containing adapter proteins and the Ras exchange factor Sos, as well as from serpentine receptors via the beta gamma subunits of heterotrimeric G proteins and the Ras exchange factor Ras-GRF (or Cdc25). Proteins that can stimulate the GTPase activity of Ras (GAPs) ensure that following mitogenic stimulations, they return to their inactive GDP-bound state; amongst these proteins are p120-GAP, neurofibomin (the product of the susceptibility gene to type I neurofibromatosis), as well as the inositol 1,3,4,5-tetrakisphosphate-dependent GAPIP4BF. Several effectors have been identified that mediate the biological effects of Ras. The serine/threonine kinase Raf-1, as well as the closely related protein B-Raf, elicit the ERK cascade of MAP kinases. Phosphatidylinositol-3-OH kinase is involved in the activation of the Rac/Rho family proteins that play a role in the control of actin polymerisation, as well as in growth control, RalGDS, RGL and Rlf, are responsible for the activation of the Ras-related protein Ral. Recent evidence, using effector domain mutants of Ras, demonstrates that these pathways cooperate to elicit the growth promoting effects of Ras proteins.

Cell Division↗
PubMed1999

Nuclear magnetic resonance and molecular dynamics studies on the interactions of the Ras-binding domain of Raf-1 with wild-type and mutant Ras proteins.

The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively. In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1. However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation. Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant. The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form. The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras. The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84. We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1. A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras. Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity. We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras.

Amino Acid Sequence↗