Article

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

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

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.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... 39). Expression in S. (cereiisiae of the [Asn-16]Ha-ras mutant, which possesses reduced affinity for guLanine nucleotides, was found to be lethal to wild-type cells growing at 37°C (139). This phenotype was postulated to result from the nucleotide-free species of Ras forming a dead-end complex with an exchange factor (139). ...
... Expression in S. (cereiisiae of the [Asn-16]Ha-ras mutant, which possesses reduced affinity for guLanine nucleotides, was found to be lethal to wild-type cells growing at 37°C (139). This phenotype was postulated to result from the nucleotide-free species of Ras forming a dead-end complex with an exchange factor (139). The [Asn- 16]Ha-ras lethal phenotype was shown to be suppressible by either inactivation of the CLDC25 gene (86,139). ...
... This phenotype was postulated to result from the nucleotide-free species of Ras forming a dead-end complex with an exchange factor (139). The [Asn- 16]Ha-ras lethal phenotype was shown to be suppressible by either inactivation of the CLDC25 gene (86,139). Conversely, the ((1(25-1 phenotype was itself suppressed by the expression of [Asn-161Ha-ras probably reflecting GTP bound to the mutant r(as protein (86) gene. ...
Article
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.
... Substitutions at positions 16, 28,35, 36, 38, 116, 117, 119, 144 and 147 also lead to activation. Nucleotide binding is reduced by m utating residues 116, 117, 119 and 144 so activating the protein (Clanton et al, 1986;Der et al, 1986;Feig et al, 1986;Sigal et al, 1986b;W alter et al, 1986). M utation of Lysl6 disrupts the stability of the polypeptide chain, has a steric effect on Seri 7 1 8 and reduces nucleotide affinity but not specificity since L ysl6 does not bind the guanine ring (Sigal et al, 1986b). ...
... Nucleotide binding is reduced by m utating residues 116, 117, 119 and 144 so activating the protein (Clanton et al, 1986;Der et al, 1986;Feig et al, 1986;Sigal et al, 1986b;W alter et al, 1986). M utation of Lysl6 disrupts the stability of the polypeptide chain, has a steric effect on Seri 7 1 8 and reduces nucleotide affinity but not specificity since L ysl6 does not bind the guanine ring (Sigal et al, 1986b). Phe28 binds the guanine base through aromatic-aromatic interactions and interacts w ith Lysl47. ...
Thesis
The ras gene family consists of three members that encode highly similar proteins of 21Kd (p21ras/Ras). This protein is plasma membrane associated, binds guanine nucleotides and has intrinsic GTPase activity. Activating point mutations render Ras insensitive to regulation by GAP (GTPase activating protein) and it remains in the active GTP bound state. Membrane association of Ras has been shown to be essential for its biological activity. The plasma membrane targeting of Ras is accomplished by a series of post-translational modifications which occur in 2 steps. Step 1 involves the CAAX motif (C = cysteine, A = aliphatic and X = any amino acid) at the C- terminus. The cysteine is alkylated by C15 farnesyl, the -AAX amino acids are removed and the new C-terminal cysteine undergoes methylesterification. Step 2 involves palmitoylation of cysteine residues near the CAAX motif in the case of H-, N- and K-ras (A). Membrane localisation of K-ras (B) appears to involve electrostatic interaction of the polybasic region (K175-180) with the membrane. Other CAAX containing proteins (rap 1A, G25K) are prenylated with a C20 geranylgeranyl moiety rather than C15 farnesyl. Geranylgeranylation of H- and K-ras (B) also leads to membrane association of the protein but specific targeting to the plasma membrane requires the presence of the polybasic domain or the palmitoylation sites. Another family of proteins (p60src, Gag, cytochrome b5 reductase) is membrane associated by the addition of myristic acid to the N-terminus. Myristoylation can also allow Ras proteins to be membrane associated but specific plasma membrane targeting remains dependent on the presence of palmitoylation sites or a polybasic region. Upstream of the CAAX motif is the hypervariable domain - a region that shows less than 20% homology between the ras genes. The function of this domain is not known and it may simply connect the N- and C-termini. However this region could also confer specificity on the interaction of different Ras proteins with different effector and/or regulatory proteins. Deletions within this region destroy transforming ability and reduce MAP kinase activity suggesting that effector interaction is disrupted. N17 deletion mutants rescue proliferation of NIH 3T3 cells indicating that exchange factor interaction is also influenced by the hypervariable region. This thesis attempts to establish a relationship between the biological activity of Ras, its cellular location and post-translational processing events. A functional role for the hypervariable domain is also examined.
... The P-loop/G-1 motif is one of the most highly conserved regions in nucleotide binding proteins, including both GTPases and protein kinases (59), and is involved in binding to the β-phosphate and Mg 2+ in the nucleotide binding pocket. Mutation of this conserved lysine in different nucleotide binding proteins has been shown to result in decreased affinity for nucleotides (60), loss of enzymatic activity (61), or both (62). In murine ARL13B this region is G 28 LDNAGK 34 . ...
... We initially used K34A to test whether autophosphosphorylation occurred on mouse ARL13B as this residue in the P-loop is perhaps the most highly conserved residue in ATP-and GTP-binding proteins and its mutation typically results in loss of nucleotide binding and/or enzymatic activities (59,60,82). We found ARL13B[K34A] expressed and purified like the wild type protein despite its weaker binding to mant-Gpp(NH)p (Table III), as predicted. ...
Article
Full-text available
Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert Syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ARF family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of ~20 kDa and at least one key residue difference in the consensus GTP binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine ARL13B protein. We report results from assays for solution-based nucleotide binding, intrinsic and GAP-stimulated GTPase, and ARL3 GEF activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine ARL13B is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.
... R15 exhibited high nanomolar affinity for the apo state of all three RAS isoforms but undetectable binding to GTP or GDP-loaded RAS (Figures 1B and C). When expressed in cells as a fusion with cyan fluorescent protein (CFP), R15 preferentially interacted with HRAS(K16N) over wild type (WT) and HRAS(G12V), consistent with HRAS(K16N) residing in the apo state (Sigal et al., 1986; Figure S2A). Furthermore, R15 bound HRAS(D119N), which has reduced affinity for nucleotide (Cool et al., 1999). ...
Article
Full-text available
RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.
... In EML4-ALK-expressing cells, we observed substantial enrichment of the RAS-GTP reporter at EML4-ALK cytoplasmic protein granules and not at the PM ( Figures 3E and 3F). Co-expression of a dominant-negative RAS (RASN17) (Sigal et al., 1986) that interferes with RAS activation (GTP-loading) decreased colocalization of the RAS-GTP reporter at EML4-ALK granules, as did introduction of mutations into the RBD component of the GFP-RBD reporter (RBD R59A/ N64D) that decrease affinity for RAS-GTP (Biskup and Rubio, 2014) ( Figure 3F). The collective findings show that local RAS activation and accumulation of RAS-GTP occurs at membraneless EML4-ALK cytoplasmic protein granules. ...
Article
Full-text available
Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.
... Next, we genetically interfered with Ras activity using dominant negative mutant Ras construct (H-Ras, S17N) [29][30][31]. This mutant H-Ras competes with endogenous wild type H-Ras for upstream activators but cannot activate downstream effectors [31,32]. ...
Article
Full-text available
Several studies suggest that soluble Amyloid β (Aβ) oligomer-induced aberrant neuronal cell cycle re-entry is the initial trigger for a significant part of the neuronal degeneration and loss in Alzheimer's disease (AD). In this study, we investigated the role of Ras, which is a well-known proto-oncoprotein, in soluble Aβ oligomer- induced aberrant neuronal cell cycle activation and subsequent cell loss using retinoic acid differentiated human SH-SY5Y neuroblastoma cells as model system. In line with previous literature, we showed that in vitro preparations of soluble Aβ42 oligomers triggered cell cycle activation but not cell proliferation. As a new finding, we showed that Farnesylthiosalicylic acid (FTS), a specific chemical Ras inhibitor, prevented soluble Aβ42 oligomer preparationinduced cell cycle activation. Moreover, we showed that the expression of dominant negative mutant H-Ras (S17N) prevented soluble Aβ42 oligomer preparation-induced cell cycle activation, confirming the specific role of Ras in this pathway. As a possible better mimic of the situation in the AD brain, we prepared soluble oligomers from Aβ42 : Aβ40 (3:7) peptide mixture and showed that this oligomer preparation similarly induced cell cycle activation which was also inhibited by the Ras inhibitor. Finally, we showed that FTS prevented soluble Aβ42 oligomer preparation-induced cell death in our retinoic acid differentiated SH-SY5Y cells. Overall, our results strongly suggest that Ras activity is required for soluble Aβ oligomer-induced aberrant neuronal cell cycle re-entry and subsequent neuronal loss, which are considered important mechanisms in AD pathogenesis.
... In an effort to elucidate the function of Dexras1, we selectively introduced an Ala 3 Val mutation in codon 178, based on structural analogy to a known activating mutation in H-Ras (H-Ras[A146V]; Ref. 43). This mutation in the ⌺4 region of Dexras1 is predicted to interrupt the guanyl nucleotide-binding pocket, resulting in decreased affinity of the mutant for both GTP and GDP, as well as an increase in the exchange rate of GDP for GTP (43,44). Guanyl nucleotide exchange, particularly the release of GDP, is the rate-determining step in the physiological activation of most G proteins (45). ...
Article
Dexras1 is a novel GTP-binding protein that shares structural similarity with the Ras family of small molecular weight GTPases and is strongly and rapidly induced during treatment with dexamethasone. The function of Dexras1 and its contribution to glucocorticoid-dependent signaling in the corticotroph cell are unknown. The present study was undertaken to examine the potential role of Dexras1 in the regulation of peptide hormone secretion in the AtT-20 corticotroph cell line. To determine the effects of Dexras1 expressed independently of glucocorticoid treatment, expression plasmids for wild-type and constitutively active mutant Dexras1 proteins were cotransfected with human GH (hGH), which provides an ectopic marker for the stimulus-coupled secretory pathway. GTP binding properties and the GTP to GDP ratio of wild-type and mutant Dexras1 proteins were examined in transiently transfected AtT-20 and COS-7 cells. Stimulated and constitutive components of secretion were assessed after 2-h incubations with 5 m...
... elongation factor Tu (EF-Tu) [37], elongation factor G [38], Ha-Ras [39]} prompted us to substitute these two amino acids. Corresponding mutations in the Ha-Ras protein [40] and in EF-Tu [41] have already been demonstrated to affect guanine-nucleotide binding. For this purpose, the hypB gene was cloned into phage MI3 and mutated at these positions to create asparagine codons. ...
Article
The product of the hypB gene, which is required for the maturation of the three [NiFe]hydrogenases of Escherichia coli, is a member of the GTPase family and exhibits a low intrinsic GTPase activity. It was studied whether or not GTP hydrolysis by HypB is coupled to nickel insertion into hydrogenases and to maturation of hydrogenases. Mutations were introduced into the hypB gene at sites expected to code for amino acids involved in guanine-nucleotide binding. Lys117 of G-motif 1, as well as Asp241 of G-motif 4 were substituted by asparagine residues. The purified mutant HypB proteins showed strongly reduced, but still significant, GTPase activity. In the case of [D241N]HypB, the kcat/Km value was lowered by a factor of 85 and the specificity of the enzyme for GTP was apparently lost, with other nucleoside triphosphates including XTP becoming compatible substrates. The decrease in GTPase activity was even more pronounced for [K117N]HypB. To assess the functionality of these HypB proteins in vivo, the wild-type hypB gene in the chromosome of E. coli was replaced by the mutant alleles. The resulting mutant strains BKN117 and BDN241 were affected in hydrogen metabolism under fermentative conditions. BKN117 did not display hydrogenase activity due to a loss of nickel incorporation into the large subunit. BDN241 exhibited a reduction of hydrogenase activity by 44% and only a portion of the hydrogenase 3 large subunit was in the mature nickel-containing form. From these results, it is concluded that GTP hydrolysis catalysed by HypB is an integral process in nickel incorporation into hydrogenases.
... Site-directed mutagenesis was used to prepare a series of disease-associated mutants for each of the cloned RTKs, taking advantage of the information available in the OMIM, PubMed, VarSome and Cosmic [18] databases. Table 1 lists the available RTK mutants; the links to the associated pathologies are given in Supplementary Table 2. Kinase-dead (KD) mutants were also prepared, usually by mutating a distinct lysine residue in the catalytic loop that stabilizes the pentavalent transition state of ATP γ-phosphate [19][20][21][22] (Supplementary Table 3). The cloned RTKs were verified by sequencing and validated for expression and catalytic activity as described else where [14]. ...
Article
Full-text available
Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.
... 12,16 Mutation of the Ras P-loop K to N reduces affinities for GDP and GTP by 65-and 180-fold respectively, from 20 nm to 1.3 μM and from 10 nM to 1.8 μM. 17 Based on this work, mutation of the DSP P-loop K has been suggested to generate protein that is both binding-deficient and catalytically inactive. 12 In support of this, increasing the GTP concentration from 100 to >300 μM resulted in some GTP hydrolysis for K41A dynamin, 13 which was interpreted in terms of a lowered affinity for GTP, a lowered k cat , or both. ...
Article
Dynamin‐superfamily proteins (DSPs) are large self‐assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P‐loop of DSP GTPase domains results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant‐negative to impair DSP‐dependent processes. However, the precise deficit within the GTPase domain of the P‐loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P‐loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P‐loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely‐used DSP P‐loop K to A mutation and the basis of its dominant‐negative effects in the cell. This article is protected by copyright. All rights reserved.
... In EML4-ALK-expressing cells, we observed substantial enrichment of the RAS-GTP reporter at EML4-ALK cytoplasmic protein granules and not at the PM ( Figures 3E and 3F). Co-expression of a dominant-negative RAS (RASN17) (Sigal et al., 1986) that interferes with RAS activation (GTP-loading) decreased colocalization of the RAS-GTP reporter at EML4-ALK granules, as did introduction of mutations into the RBD component of the GFP-RBD reporter (RBD R59A/ N64D) that decrease affinity for RAS-GTP (Biskup and Rubio, 2014) ( Figure 3F). The collective findings show that local RAS activation and accumulation of RAS-GTP occurs at membraneless EML4-ALK cytoplasmic protein granules. ...
... In vitro mutagenesis studies have further shown that substitutions at position 59,63,116,117,119 and 146 can also confer transforming activity. (Fasano et al., 1984;Sigal et al., 1986a;Walter et al., 1986) although these mutations have not been found in human tumours. Further in vitro analysis has revealed that the substitution of glycine 12 ...
Thesis
Ras genes are found mutated in about 30% of all human malignancies, being one of the most frequent mutations in human cancer. They code for 21 kiloDalton GTPases that act as molecular switches at a critical crossroad in the control of proliferation, differentiation and many other cellular processes. When in its active, GTP-bound state, Ras can interact with its downstream targets or effectors to carry out its biological function. The best characterised Ras effector is the Raf serine/threonine kinase, through which Ras activates the MAP kinase cascade. The work presented in this thesis, describes the identification of Phosphoinositide 3-kinase (PI 3-kinase) as an additional Ras effector. Ras, when bound to GTP, interacts through its effector domain with the p1 10 catalytic subunit of PI 3-kinase. This interaction results in stimulation of the lipid kinase activity of PI 3-kinase, both in intact cells and in an in vitro reconstitution system. Furthermore, expression of the dominant negative mutant N17 Ras, inhibits EGF and NGF-induced activation of PI 3-kinase in intact PC 12 cells. In order to study the contribution of PI 3-kinase to Ras function, mutations in the effector domain of Ras have been generated, that selectively impair the ability of Ras to interact with different effectors. The Y40C mutation allows Ras to interact only with PI 3-kinase, T35S and D38E mutants interact only with Raf and the E37G mutant interacts only with RalGDS. These partial loss of function mutants, together with activated and dominant-negative versions of PI 3-kinase and other effectors, have been used to study the effects of the different Ras effector pathways in several cell systems. It is shown that Ras needs to activate simultaneously several pathways in order to efficiently transform fibroblasts. Although no single pathway is sufficient, PI 3- kinase makes a critical contribution to Ras transforming potential: inhibition of PI 3- kinase function inhibits loss of contact inhibition, loss of anchorage-dependence for growth and morphological transformation induced by Ras. PI 3-kinase, through the activation of the Rac GTPase, is also a critical mediator of the effects of Ras on the actin cytoskeleton. In addition, evidence is presented that activation of PI 3-kinase may play a role in protecting tumour cells harbouring Ras mutations from programmed cell death. The results suggest that the PI 3-kinase pathway may be an attractive novel target for therapeutic intervention in the treatment of tumours where Ras is involved.
... The findings with HRAS G15A were similar to those made by two earlier studies focused on mutations at two other residues in the HRAS G1 GxxxxGKS/T motif. One study found that a K16N mutation caused 100-fold reduced affinity for GDP and GTP and HRAS K16N impaired yeast growth (Sigal, Gibbs, D'Alonzo, Temeles, et al. 1986). This growth defect could be overcome by concurrent expression of activated yeast RAS2. ...
Article
Full-text available
The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated RAS genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer. Only recently, exome sequencing studies identified cancer-associated alterations in two RHO family GTPases, RAC1 and RHOA. RAS and RHO proteins share significant identity in their amino acid sequences, protein structure and biochemistry. Cancer-associated RAS mutant proteins harbor missense mutations that are found primarily at one of three mutational hotspots (G12, G13 and Q61) and have been identified as gain-of-function oncogenic alterations. Although these residues are conserved in RHO family proteins, the gain-of-function mutations found in RAC1 are found primarily at a distinct hotspot. Unexpectedly, the cancer-associated mutations found with RHOA are located at different hotspots than those found with RAS. Furthermore, since the RHOA mutations suggested a loss-of-function phenotype, it has been unclear whether RHOA functions as an oncogene or tumor suppressor in cancer development. Finally, whereas RAS mutations are found in a broad spectrum of cancer types, RHOA and RAC1 mutations occur in a highly restricted range of cancer types. In this review, we focus on RHOA missense mutations found in cancer and their role in driving tumorigenesis, with comparisons to cancer-associated mutations in RAC1 and RAS GTPases.
... En revanche, l'affinité pour les nucléotides est faible. C'est assez surprenant pour une GTPase car les affinités mesurées pour les GTPases de la famille de Ras sont de l'ordre du nM (Sigal et al., 1986) mais cette faible affinité à également été décrite pour MnmE (Scrima et Wittinghofer, 2006;Yamanaka et al., 2000) ou encore Era (Sullivan et al., 2000). Figure 49). ...
Article
Full-text available
Genome sequencing programs have revealed many genes of unknown function. The systematic disruption of these genes revealed the essentiality for some of them. Studying orphan proteins became of first importance as they are ideal targets for new antibacterial compounds. YphC is a GTPase from Bacillus subtilis that meets these criteria. It is well conserved throughout bacterial kingdom but is not found in eukaryota or archeas, strengthening the choice of this protein as a future target for antibacterial drugs. YphC has the particularity to possess two GTPase domains in tandem. As a unique protein, we decided to study YphC from a biochemical point of view to better understand its catalytic mechanism. We overexpressed and purified the protein, either wild type or mutants. We measured its enzymatic constants and characterize potassium activation effect on its hydrolytic activity. We showed that YphC displays a high GTPase activity and that GD1 bears the majority of this activity .GD2 would thus have a regulatory role in the protein. We also studied the role of YphC in vitro. We showed that the protein was able to interact with ribosome from Bacillus subtilis in a nucleotide dependant manner, suggesting that YphC plays a role in ribosome biogenesis.
Chapter
The RAS-mediated signal transduction pathway, which is highly conserved among eukaryotes, plays key roles in multiple cellular and developmental processes including cell proliferation, differentiation and migration. In recent years, a combination of biochemical and genetic studies has allow scientists to identify many key players involved in RAS-mediated signal transduction and has begun to elucidate the mechanisms of activation and regulation of this pathway (see other chapters in this book). From receptor tyrosine kinases (RTKs) to RAS to MAP kinases, the main signal transduction pathway has been well established. The connections between this pathway and several other major signal transduction pathways have also begun to be revealed.
Article
Organisms integrate information of current environmental stressors and can adjust themselves against harmful events that might occur in the future. The molecular processes that lead to such “anticipatory” behaviors, although of great interest, are mostly unexplored and the minimal genetic requirements for reconfiguring key signaling networks in order either to create or to strengthen such vital “anticipatory” capabilities is largely unknown. We identified new “anticipatory” phenotypes in yeast cells by evolving yeast strains that strongly associate a present modest stress with a future deadly one. Whole genome sequencing and classic genetic analysis revealed that two dominant negative ras2 alleles (ras2-K23N and ras2-G17C) displayed a strong “anticipatory” ability being highly resistant to oxidative stress, extremely thermotolerant and long lived only following an initial mild heat shock. We suggest that such “anticipatory” phenotypes can be easily evolved by a single point mutation in a key signaling protein, the Ras2 small GTPase, and we propose a molecular model describing how specific ras2 alleles, and not null ras2 mutants, or mutations in other components of the Ras/cAMP pathway, can enhance the “predictive ability” of cells for future lethal stressors.
Article
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.
Chapter
Within the GTPase family of proteins the members referred to as G-proteins provide a signal transduction coupling mechanism for many cell surface receptors as described in other chapters in this volume. G-proteins are responsible for regulating an intracellular effector, such as an ion channel or an enzyme, in response to an activated receptor (Johnson and Dhanasekaran 1989). G-proteins exist as heterotrimers composed of α,β, and γ subunits. The G-protein β subunit is the component that binds GDP and GTP. Receptors coupled to specific G proteins catalyze GDP dissociation, allowing GTP to bind. The GTPα complex in turn regulates the activity of specific effectors. The lifetime of the activated GTPα complex is controlled by an intrinsic GTPase encoded in the α subunit which hydrolyzes the bound GTP to GDP (αGDP) returning the a subunit to an inactive state.
Chapter
The membrane-bound ribosomes of the rough endoplasmic reticulum (RER) are engaged in the synthesis of secretory proteins, resident lumenal proteins of the exocytic and endocytic membrane systems, and the majority of cellular integral membrane proteins. The information responsible for the selective delivery of ribosomes to the RER is contained in an amino terminal signal sequence. Ribosomes synthesizing proteins with RER-specific signal sequences are subsequently targeted to a membrane-bound translocation site or “translocon.” The translocon is a multicomponent protein assembly that mediates the unidirectional transport of proteins or protein domains across the RER membrane. Transport of nascent polypeptide chains across the endoplasmic reticulum (ER) membrane has been proposed to occur through a proteinaceous transport site or channel (Gilmore and Blobel 1985; Simon and Blobel 1991) that may consist of several integral membrane proteins which have been identified by cross-linking to nascent polypeptides (Krieg et al. 1989; High et al. 1991; Kellaris et al. 1991; Görlich et al. 1992). Upon entry into the RER lumen, the nascent polypeptide undergoes modifications and folding reactions that result in the assembly of a mature protein. The focus of this article will be upon the roles of signal recognition particle (SRP) and SRP receptors, two multisubunit GTP-binding proteins that mediate the initial phases of the protein translocation reaction.
Chapter
Dynamin is a 100-kDa polypeptide initially identified in brain tissue by virtue of its copurification with microtubules (Shpetner and Vallee 1989). Molecular cloning and biochemical analysis revealed dynamin to be a GTPase (Obar et al. 1990; Shpetner and Vallee 1992). Identification of a possible homologue of dynamin in Drosophila together with recent transfection studies with mutant forms of rat dynamin have indicated an important role for the protein in the initial steps of the endocytic pathway.
Chapter
ras proteins belong to a superfamily of guanine nucleotide binding proteins that cycle between the active GTP- and inactive GDP-bound states (Bourne et al. 1991). They become activated by exchanging GTP for bound GDP, a process catalyzed by a guanine nucleotide exchange factor (GEF) (also called ras-GRF, guanine nucleotide releasing factor) (Shou et al., in press). They become deactivated by hydrolyzing GTP to GDP, which is catalyzed by GTPase activating proteins, ras-GAP and NF-1 (Bollag and McCormick 1991). Three ras genes, H-, N-, and K-ras, code for extremely similar 21-kDa proteins (p21s) that are localized to the inner surface of the plasma membrane. A role for p21 in the control of cell proliferation was first suggested by the observation that mutant ras genes are responsible for the oncogenicity of a set of rodent retroviruses. Subsequently, it was shown that these same mutations, which lock ras proteins in the active GTP-bound form, are present in endogenous ras genes from a wide variety of human tumor types (Barbacid 1987). Moreover, expression of the mutated genes in tissue culture cells or in transgenic animals promotes a neoplastic phenotype (Quaife et al. 1987).
Article
The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.
Article
A new family of highly conserved genes, designated rho, has recently been isolated and characterized (P. Madaule and R. Axel, Cell 41:31-40, 1985). These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rats, and humans, and their 21,000-dalton products are highly homologous. The rho p21 protein shares 35% amino acid homology with the Harvey ras p21 protein and on this basis has been proposed to be a G protein. We expressed the Aplysia californica rho gene in Escherichia coli and purified its p21 protein to more than 90% purity. The availability of the rho protein in high quantities made it possible to establish its high affinity for guanine nucleotides. The rho p21 protein had nucleotide-binding properties similar to those of the ras p21 protein. However, a comparison of these proteins revealed some important differences regarding their specificities and affinities. Finally, the rho p21 protein had GTPase activity almost identical to that of a normal ras p21 protein, the rates being 0.106 and 0.105 mol/min per mol of p21, respectively. Thus, the results suggest that the degree of homology found between the ras and rho genes products most likely is related to the conservation of sequences relevant to their ability to bind and hydrolyze guanine nucleotides. The fact that the rho p21 protein binds and hydrolyzes GTP strongly suggests that it is a G protein with a potential regulatory function conserved in evolution.
Chapter
Are studies by analogy fruitful to understanding protein structure and function? With protein sequences accumulating at an astonishing rate through gene cloning and DNA sequencing, a great deal of protein structure and function can be learned by analogy with members of the superfamily whose structures have been determined and whose molecular mechanisms of action are known. The best case in point, perhaps is in unraveling the elusive function of ras p21. The molecular model of p21 has been constructed by analogy with the crystal structure of the E. coli elongation factor, EF-Tu (McCormick et al., 1985; Jurnak, 1985). This p21 model is remarkably consistent with the actual three dimensional structure of p21 later determined by X-ray crystallography (De Vos et al., 1988). The recent identification of the GAP protein which stimulates GTPase activity of p21 (Trahey and McCormick, 1987), is a conceptual offspring of similar biochemical mechanism well understood for the function of EF-Tu in protein synthesis (Kaziro, 1978). Furthermore, analogy with the well characterized G-proteins, which regulate transmembrane cell signalling in the adenylate cyclase systems and light transduction in retina, forms the foundation for the current belief that ras p21 mediates transmission of growth signals to their intracellular effectors that control cell proliferation and differentiation (Bourne and Sullivan, 1986).
Chapter
In mammalian cells, the adenylate cyclase is regulated by extracellular signaling molecules, hormones and neurotransmitters. This regulation involves specific transmembrane receptors and transducers which are heterotrimer G-proteins. In response to the liganded receptor the G-protein is activated by dissociation of the GTP-bound a subunit which can then activate the adenylate cyclase. Although this complex system which also involves negative regulatory circuit, has been extensively studied, the details of its functioning have not yet been completely elucidated.
Chapter
A milestone in the quest to understand the molecular basis of human cancer was the recognition that proto-oncogenes altered by somatic mutation might act in a dominant fashion in cellular transformation. Such a process may involve illegitimate recombination of proto-oncogenes with distant genomic sequences through chromosomal rearrangement resulting in formation of a novel gene-fusion product, the prototypic examples being the 210- and 190-kDa bcr-abl tyrosine kinases in Philadelphia-positive chronic myeloid and acute lymphoblastic leukaemias [1] or deregulation of proto-oncogene expression, for example, that of c-myc when juxtaposed with the immunoglobulin gene loci in the reciprocal translocations which characterize Burkitts’ lymphoma [2]. Alternatively, overexpression may result from an increase in gene copy number (amplification). With few exceptions, the most striking being those of N-myc and erb-B2 in childhood neuroblastoma [3] and breast cancer [4], respectively, proto-oncogene amplification is consistently detected in few human cancers and may reflect a relatively late event in tumour evolution.
Chapter
A large number of transmembrane signalling systems transduce signals through heterotrimeric GTP binding proteins (G-proteins). In the most intensively studied system -adenylate cyclase, there are two distinct G-protein transducing systems which respond to stimulatory and inhibitory receptors respectively. The studies on the hormonally regulated adenylate cyclase have led to a detailed understanding of the molecular mechanism of signal transduction. This system therefore serves also as an archtype model for the study of other transmembrane signalling systems which possess heterotrimeric GTP binding proteins as transducer elements. One system in which the molecular mechanism of G-protein transduction is not known is the proliferation signal activated by the monomeric G-protein p21RAS. The involvement of a RAS protein as a transducer in the activation in the yeast S. cerevisiae adenylate cyclase opens new avenues towards the understanding of RAS function in mammalian cells.
Chapter
The Discovery that the viral oncogenes carried by the Harvey (v-H-ras) and Kirsten (v-K-ras) sarcoma viruses were transduced versions of normal cellular genes supported the idea that alterations in certain key cellular genes (cellular H-, K-, and N-ras) could contribute to human carcinogenesis (Ellis et al. 1981; Chang et al. 1982b). The identification of transforming versions of human ras genes in DNA isolated from human tumor cells in gene transfer assays confirmed this possibility (Der et al. 1982; Parada et al. 1982; Santos et al. 1982; Shimizu et al. 1983). Molecular cloning and analyses of these tumor-derived ras sequences quickly established that conversion of the normal genes to the oncogenic versions was due simply to single amino acid substitutions, primarily at residues 12 (Tabin et al. 1982; Reddy et al. 1982; Taparowsky et al. 1982), 13 (Bos et al. 1985), and 61 (Yuasa et al. 1983). Concurrent with the development and application of sensitive molecular techniques for the detection of genetic point mutations in tumor DNAs was the finding that oncogenic ras sequences were frequently associated with a broad spectrum of human malignancies (reviewed in Bos 1988, 1989).
Chapter
The ras genes are of three varieties, each with a similar exon-intron structure and each encoding a protein of 21 kilodaltons (kD): p21. The Harvey ras oncogene was the first to be directly implicated in human neoplasia. An H-ras gene from a bladder carcinoma cell line was isolated and characterized by its ability to transform the murine cell line NIH 3T3. Single-base-pair differences at specific codons distinguish normal pvoto-ras genes from their oncogenic counterparts. These changes translate to single amino acid substitutions in the mutant p21 products. In addition, elevated levels of normal p21, corresponding in some cases to amplification of the ras gene, have been associated with neoplasia. The p21 protein appears to be similar to the G proteins that act as second messengers, possibly in the phosphatidyl inositol (PI) system, although the latter has not yet been conclusively proved. Normal p21 has GTPase activity that converts the protein from an active to an inactive form. Mutant p21s may have reduced or ineffectual GTPase activity leading to constitutive activation of the protein. Activated protein may transduce a signal(s) important for cell growth; thus constitutive activation may lead to misregulated growth. The ras genes are widely conserved among animal species and have been found in yeast as well. Yeast ras genes bear some homology to vertebrate ras genes and have been shown to be of importance to yeast growth and cell cycle. Mutant ras genes and proteins can be experimentally distinguished from their normal counterparts by specific DNA probes and antibodies. These and similar reagents may eventually prove to be of importance in cancer diagnostics and therapeutics.
Article
While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.
Article
CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.
Article
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.
Article
Full-text available
The signal recognition particle (SRP) receptor (SR) is a heterodimer of two polypeptides (SRα and SRβ) that each contain a GTP-binding domain. The GTP-binding domain in the peripheral membrane SRα subunit has a well defined role in regulating targeting of SRP-ribosome-nascent chain complexes to the translocon. The only well established function for the transmembrane SRβ subunit is anchoring SRα on the endoplasmic reticulum membrane. Deletion of the amino-terminal transmembrane domain of SRβ did not affect receptor dimerization, but revealed a cryptic translocation signal that overlaps the GTPase domain. We demonstrate that the domain of SRα that binds SRβ does so by binding directly to the nucleotide-bound form of the GTPase domain of SRβ. An SRβ mutant containing an amino acid substitution that allows the GTPase domain to bind XTP dimerized with SRα most efficiently in the presence of XTP or XDP, but not ATP. Our results suggest an additional level of regulation of SRP receptor function based on regulated dissociation of the receptor subunits.
Article
Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.
Article
In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25. We isolated several dominant extragenic suppressors of the yeast cdc25 mutation. They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35). One of these suppressors was a mutated RAS2 gene in which the transition C/G----T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein. The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L.A. Feig, B. Pan, T.M. Roberts, and G.M. Cooper, Proc. Natl. Acad. Sci. USA 83:4607-4611, 1986). These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.
Article
The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.
Article
Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae. An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25. Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells. The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS. These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.
Article
We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.
Article
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.
Article
Full-text available
A novel method for the analysis of putative G-proteins has been developed that reveals the existence of a large family of GTP/GDP-binding proteins with similar characteristics to those of p21ras in 3T3 cell plasma membranes. In the presence of Mg²⁺, exchange of [α-³²P]GDP with prebound ligand was very slow, but, as with p21ras, exchange was dramatically accelerated by excess EDTA. In the presence of Mg²⁺, three classes of binding sites were distinguishable. However, no p21ras was detected in the membranes with the pan-reactive anti-ros antibody, Y13-259. Gel filtration analysis resolved two peaks of binding activity centering at 60 and 21 kilodaltons. High resolution anion exchange chromatography separated at least 11 unique GDP-binding proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized membranes, none of which cross-reacted with a pan-Gα antiserum. Analogous to p21ras, the binding activities of 9 of the 11 species were sensitive to the thiol reagent N-ethylmaleimide, and six peaks possessed detectable GTPase activity in the absence of extrinsic factors. Addition of cytosol activated the GTPase activity of four of the peaks. We infer that the 11 peaks represent novel, small molecular weight guanine nucleotide-binding proteins, similar to those recently described in brain membranes.
Article
We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.
Article
We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.
Article
We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.
Article
Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.
Article
Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.
Thesis
Most testicular germ cell tumours (TGCTs) have a modal chromosome number in the hypotriploid range and and tumour progression is thought to result from net loss of chromosomes from a near tetraploid carcinoma in situ cell. Around 1.5% of TGCTs occur in a familial form thus suggesting a possible role for tumour suppressor genes in testicular tumorigenesis. To find out whether particular areas of the TGCT genome show marked loss of heterozygosity (LOH) allele loss studies were carried out on tumours from 41 patients using a number of polymorphic ezyme and DNA RFLP markers covering 23 chromosome arms. In addition aneuploid cultures were successfully established from seven cases and the results from these cultures were compared with those from corresponding tumours. LOH at a frequency of >10% was found on the following chromosome arms; 3p (2 losses out of 18 informative individuals), 3q (1/8), 4 (1/10), 5q (6/33), 11p (4/13), 11q (4/24), 12p (1/7), 13q (3/18), 16p (3/24) and 18p (1/5). LOH at a fequency of <10% was found on 1p (2/37), 1q (3/34), 5p (1/30), 7p (1/27), 7q (2/22), 9q (1/19), 14q (2/22), 15 (1/23), 18q (2/25) and 20 (1/26). No losses were found on 6p (six informative individuals), 12q (33) and 17p (23). Additional losses on 1p, 1q, 3p, 9q, 11p, 11q, 14q, 18p and 18q were observed in the tumour cultures. The relatively high frequency of allele loss on 3p, 5q, 11p, 11q, 13q and 16p may indicate the presence of tumour suppressor genes on these chromosome arms which are important in the development of TGCTs. The extent of allele loss as defined by fractional allele loss was not found to be significantly different between the two histologic subtypes seminoma and nonseminoma. An examination of allele intensity in tumour DNA has demonstrated allelic evenness in over 70% of tumours for probes on 1p36, 3q, 4, 5p, 5q, 7q, 11p, 11q, 12q14, 13q and 15q. The only chromosome arm demonstrating an uneven pattern in more than 70% of tumours is 12p. These findings suggest that the changing pattern of genomic organization occuring during tumour progression is non-random. An isochromosome for the short arm of chromosome 12 is a highly specific cytogenetic abnormality found in the majority of TGCTs. The retention of heterozygosity for all informative markers on 12q suggests that formation of the i(12p) markers occurs following polyploidization. Mutations in the p53 gene which is located on 17p are considered to be among the most common genetic alterations in human cancers. The retention of heterozygosity for all informative 17p loci suggests that alterations in the p53 gene are not important for the predisposition and development of TGCTs. Consistent with this finding is the lack of any consistent change in p53 mRNA levels in TGCTs. Inactivation of the retinoblastoma (RB) tumour suppressor gene is associated with the development of several human malignancies. An investigation of RB mRNA revealed decreased levels in all TGCTs analysed but no alterations in transcript size. Rearrangemet of the RB gene was demonstrated in a single TGCT. These findings suggest regulation of transcription and/or transcript half- life or gene mutations may be responsible for the decreased RB mRNA levels in human TGCTs. Analysis of the H-ras oncogene mRNA revealed decreased levels in nearly all tumours examined. The reduced levels did not always coincide with LOH at this locus thus suggesting a decrease in the level of transcription and/or half-life of H-ras mRNA. There was no consistent change in the levels of N-ras and MCC40 mRNA levels.
Thesis
The Ras family of GTP-binding proteins has been implicated in the control of cellular growth, differentiation and motility. They are molecular switches which are active when bound to GTP and inactive when GDP-bound. Dictyostelium cells express five different Ras family members. Ras activation is mediated by guanine nucleotide exchange factors (RasGEFs) which facilitate exchange the of GDP for GTP by Ras proteins. The identification and molecular genetic characterisation of novel Dictyostelium RasGEFs is the main subject of this thesis. The discovery of a physiological function for the Dictyostelium RasD protein is also described. I have identified five partial cDNAs from the Tsukuba Dictyostelium cDNA project which have strong sequence homology to known RasGEFs. To investigate the function of these proteins, Dictyostelium cells with single gef gene disruptions were generated by homologous recombination. Four of the five mutants appeared to grow and develop normally but one mutant, gefB-, showed growth and developmental abnormalities. gefB- cells are unable to proliferate in axenic culture and are impaired in phagocytosis and fluid-phase endocytosis. Conversely, gefB- cells show an enhanced rate of migration, moving twice as fast as wild-type cells. The mutant cells have abnormal morphology; they are highly polarised, have many elongated microspikes and have an absence of pinocytic crowns on the cell surface. In addition, gefB- cells show a cell autonomous impairment in multicellular development. Although the phenotype of vegetative gefB- cells is similar to that of rasS- cells, it was not possible to show a direct interaction between RasS and GefB. However, GefB does exhibit the catalytic properties of a RasGEF in vivo, since it is able to complement the temperature sensitive cdc25-5 S.cerevisiae mutant. The Dictyostelium RasD protein is a small GTP-binding protein closely related to the mammalian Ras proteins Ha-, Ki- and N-Ras, and is maximally expressed during the multicellular stage of Dictyostelium development. Previous work had predicted that RasD was essential for correct differentiation and pattern formation in Dictyostelium aggregates. To further investigate the function of RasD, Dictyostelium cells containing a disrupted rasD- gene were generated by homologous recombination. Surprisingly, rasD- cells proliferate, aggregate and develop indistinguishably from wild-type cells. However rasD slugs exhibit a clear defect in phototaxis and thermotaxis exhibiting an approximately thirty-fold decrease in the efficiency of orientation towards a light or heat source relative to wild-type slugs. RasD is the first signalling protein shown to be necessary for phototaxis in Dictyostelium slugs.
Article
Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.
Article
In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.
Article
Full-text available
The ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation. Viral Harvey ras p21, cloned into Escherichia coli HB101 lambda/pRAS1, has been purified to homogeneity by a three-step procedure. The purified E. coli p21 can be bound by a monoclonal antibody to viral Harvey ras p21 and has an amino acid composition consistent with that predicted from its DNA sequence. Purified E. coli p21 has been shown by HPLC analysis on an ion-exchange column to contain near stoichiometric amounts of GDP. This noncovalently associated GDP is seen in the UV absorption spectrum of the purified protein. The noncovalently associated GDP acts as a competitive inhibitor of the interaction of added guanine nucleotides with p21.
Article
Full-text available
Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.
Article
Full-text available
The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.
Article
Full-text available
The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.
Article
Full-text available
An examination of the available amino acid sequences of GTP-binding proteins has revealed that each contains a polypeptide essentially homologous for all of them. These sequences for elongation factor-Tu (EF-Tu) and the human bladder protein p21 exhibit a singular degree of homology (50%). Chemical and structural evidence indicates that this sequence in EF-Tu constitutes part of the nucleotide-binding site. The homologous sequences may therefore contribute to the GTP-binding sites of the other proteins.
Article
Proteins which bind guanine nucleotides are found in a diverse set of key regulatory positions. They are involved in hormone action, visual transduction, protein synthesis and microtubule assembly. In addition to their ability to bind guanine nucleotides these proteins possess several other common features. (i) They all have similar subunit composition, (ii) they can be ADP-ribosylated, (iii) their conformation changes depending on the nucleotide bound. These regulatory G-proteins have close functional homologies. Do they form a general class of regulatory proteins, like the protein kinases? Do they have a common evolutionary ancestry?
Article
The binding positions of ATP and AMP in pig muscle adenylate kinase (EC 2.7.4.3) have been located by X-ray diffraction analysis. For this purpose crystals have been soaked with solutions containing substrates and substrate analogues. Two adenosine pockets and the region of the phosphates have been identified. In combination with other experimental data the pockets have been assigned to the AMP site and the ATP site, respectively. Moreover, the results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation B being related to the free enzyme E and conformation A being related to E∗, the enzyme species after a substrate-induced conformational change.
Article
We have recently identified a 21,000-dalton protein, p21, coded for by Kirsten or Harvey murine sarcoma virus. On the basis of the results obtained with the p21 of a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, we concluded that the p21 was required for the maintenance of transformation induced by either virus. We report herein that when extracts from cells transformed by Kirsten or Harvey sarcoma virus are incubated with [(3)H]GDP or [alpha-(32)P]GTP, picomole quantities of guanine nucleotide can be immunoprecipitated with antisera that contain antibodies to the p21. Previously we have shown that the immunoprecipitability of [(35)S]methionine-labeled p21 of the temperature-sensitive mutant of Kirsten sarcoma virus is thermolabile. The binding of guanine nucleotide is shown herein also to be thermolabile in extracts of cells transformed by the same mutant. However, the immunoprecipitability of the [(35)S]methionine-labeled p21 in such extracts of the temperature-sensitive mutant can be preserved if the extracts containing labeled p21 are incubated with added GDP or GTP prior to heating. The results suggest an interaction between p21 and certain guanine nucleotides, and the possible roles of guanine nucleotides and p21 in the maintenance of transformation are discussed.
Article
Yeast spores lacking endogenous RAS genes will not germinate. If such spores contain chimeric mammalian/yeast RAS genes or even the mammalian H-ras gene under the control of the galactose-inducible GAL10 promoter, they will germinate in the presence of galactose and produce viable haploid progeny dependent on galactose for continued growth and viability. These results indicate that the biochemical function of RAS proteins is essential for vegetative haploid yeast and that this function has been conserved in evolution since the progenitors of yeast and mammals diverged.
Article
The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.
Article
The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.
Article
In these experiments we demonstrate that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides. These results confirm the prediction of earlier genetic and biochemical data and for the first time provide a complete biochemical assay for RAS protein function. Furthermore, we observe a biochemical difference between the RAS2 and RAS2val19 proteins in their ability to activate adenylate cyclase after preincubation with GTP.
Article
A 2.7 angstrom resolution x-ray diffraction analysis of a trypsin-modified form of the Escherichia coli elongation factor Tu reveals that the GDP-binding domain has a structure similar to that of other nucleotide-binding proteins. The GDP ligand is located at the COOH-terminal end of the beta sheet and is linked to the protein via a Mg2+ ion salt bridge. The location of the guanine ring is unusual; the purine ring is located on the outer edge of the domain, not deep within a hydrophobic pocket. The amino acids from Pro10 to Arg44 and from Gly59 to Glu190 have been assigned to the electron density with computer graphic techniques, and the resulting model is consistent with all known biochemical data. An analysis of the structure reveals that four regions of the amino acid sequence that are homologous with the family of ras oncogene proteins, termed p21, are located in the vicinity of the GDP-binding site, and most of the invariant amino acids shared by the proteins interact directly with the GDP ligand.
Article
Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.
Article
Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.
Article
Human Ha-ras1 cDNAs encoding normal and activated p21 polypeptides have been efficiently expressed in Escherichia coli and the biochemical activities associated with each polypeptide compared. In addition to the guanine nucleotide binding activity, normal p21 displays a GTPase activity which is selectively impaired by a mutation which activates its oncogenic potential.
Article
The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val12] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.
Article
Ha-ras is a member of a multigene family in man which encode highly related proteins of 189 amino acids (p21). In vitro, ras proteins bind GTP, and p21 mutants with treonine at position 59 autophosphorylate at that residue. Mutation (at amino acids 12 or 61) and elevated expression of ras genes result in cell transformation in culture, and are also observed in many types of human tumours. Normal and mutant transforming ras proteins show no differences in localization, lipidation or GTP binding. However, mutations at position 12 in recombinant (Thr 59) p21 molecules were observed to affect autophosphorylation. We have expressed the full-length normal and T24 transforming (Gly----Val at position 12) Ha-ras proteins in Escherichia coli and have purified them to homogeneity (ref. 19 and M.G. et al., in preparation); these proteins bound GTP with approximately molar stoichiometry and with an affinity comparable to partially purified mammalian proteins. Microinjection of the T24 protein into quiescent rodent fibroblasts resulted in a rapid alteration in cell morphology, stimulation of DNA synthesis and cell division; in contrast, little response was observed with the normal protein. We now report that the normal ras protein has an intrinsic GTPase activity, yielding GDP and Pi. In contrast, the T24 transforming protein is reduced 10-fold in this activity. We suggest that this deficiency in GTPase is the probable cause for the transforming phenotype of the T24 protein.
Article
In earlier studies, we molecularly cloned a normal cellular gene, c-rasH-1, homologous to the v-ras oncogene of Harvey murine sarcoma virus (v-rasH). By ligating a type c retroviral promotor to c-rasH-1, we could transform NIH 3T3 cells with the c-rasH-1 gene. The transformed cells contained high levels of a p21 protein coded for by the c-rasH-1 gene. In the current studies, we have purified extensively both v-rasH p21 and c-rasH p21 and compared the in vivo and in vitro biochemical properties of both these p21 molecules. The p21 proteins coded for by v-rasH and c-rasH-1 shared certain properties: each protein was synthesized as a precursor protein which subsequently became bound to the inner surface of the plasma membrane; each protein was associated with guanine nucleotide-binding activity, a property which copurified with p21 molecules on a high-pressure liquid chromatography molecular sizing column. In some other properties, the v-rasH and c-rasH p21 proteins differed. In vivo, approximately 20 to 30% of v-rasH p21 molecules were in the form of phosphothreonine-containing pp21 molecules, whereas in vivo only a minute fraction of c-rasH-1 p21 contained phosphate, and this phosphate was found on a serine residue. v-rasH pp21 molecules with an authentic phosphothreonine peptide could be synthesized in vitro in an autophosphorylation reaction in which the gamma phosphate of GTP was transferred to v-rasH p21. No autophosphorylating activity was associated with purified c-rasH-1 p21 in vitro. The results indicate a major qualitative difference between the p21 proteins coded for by v-rasH and c-rasH-1. The p21 coded for by a mouse-derived oncogenic virus, BALB murine sarcoma virus, resembled the p21 coded for by c-rasH-1 in that it bound guanine nucleotides but did not label appreciably with 32Pi. The forms of p21 coded for by other members of the ras gene family were compared, and the results indicate that the guanine nucleotide-binding activity is common to p21 molecules coded for by all known members of the ras gene family.
Article
More than 10 different dominant transforming genes (oncogenes) have been identified in human tumours. A human bladder carcinoma oncogene, closely related in sequence to retroviral transforming genes, is split into four exons; the first encodes the N-terminal 37 residues of p21, a protein of unknown function. The oncogene is activated by a single point mutation (guanine to thymine) resulting in the change glycine to valine at position 12 of p21 (refs 3, 4). We report here that the amino acid sequence surrounding this residue is highly homologous to the beta-subunit of mitochondrial and bacterial ATP-synthase in the region of the polypeptide that is believed to contribute to nucleotide binding. Thus, p21 may form part of an enzyme that uses purine nucleotides in catalysis. This is consistent with the finding that an equivalent murine oncogene product binds GTP.
Article
The oncogenes of Harvey and Kirsten murine sarcoma viruses (v-rasH and v-rasK) and their cellular homologues (c-rasH and c-rasK) constitute two members of the ras gene family. Each functional member of the ras gene family encodes a 21,000 molecular weight protein (p21ras). ras genes have been detected in a wide variety of vertebrate species, including Xenopus laevis (R. E. Steele, personal communication), and in Drosophila melanogaster. We report here the detection of ras-related genes in the yeast Saccharomyces cerevisiae, and the isolation of two ras-related molecular clones, c-rassc-1 and c-rassc-2, from the DNA of Saccharomyces. Both c-rassc-1 and c-rassc-2 hybridize specifically to probes prepared from mammalian ras DNA. Sequencing of c-rassc-1 reveals extensive amino acid homology between the protein encoded by c-rassc-1 and the p21 encoded by c-rasH. Our studies suggest that these clones can be used to elucidate the normal cellular functions of ras-related genes in this relatively simple eukaryotic organism.
Article
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Article
We present a genetic analysis of RAS1 and RAS2 of S. cerevisiae, two genes that are highly homologous to mammalian ras genes. By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes. However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth. We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles.
Article
Cells of the yeast, Saccharomyces cerevisiae, containing disruptions of either of two genes that are members of the rasoncogene family are viable, but haploid yeast spores carrying disruptions of both genes fail to grow. The data presented demonstrate that RAS(sc)1 and RAS(sc)2 encode an essential function in yeast. The viability of yeast cells containing disruptions of either of these genes indicates that the function(s) specified by either is sufficient to support both mitotic and meiotic cell division cycles, but that at least one wild-type gene is required for viability. There is no apparent correlation between the rates of spore germination or growth and the disruption of either RAS gene.
Article
Using an E. coli expression-vector system we have efficiently produced, purified, and characterized the full-length, nonfused, protooncogenic and oncogenic (T-24) forms of the human H-ras gene product. These purified ras proteins have been introduced by microinjection into a variety of somatic cells in an effort to examine their function. Within several hours after injection of the oncogenic form of the human H-ras protein into quiescent cells, we observe dramatic morphological changes followed by transient proliferation of the cells. In contrast, microinjection of the normal, protooncogenic form of the ras protein at the same level appears to have only little effect on the cells. Additional experiments indicate that the effect of the ras protein requires entry into the cells, is temporary, is inhibited by cycloheximide or actinomycin D, and is seen only in established cell lines. This experimental approach demonstrates that the bacterially derived and purified human H-ras proteins retain their ability to function when put back into mammalian cells and furthermore, provides a novel assay for transformation induced in established cells by the human H-ras oncogene protein.
Article
High-level expression of the p21 protein product of the BALB murine sarcoma virus v-ras gene (similar to the product of the Harvey murine sarcoma virus v-Ha-ras gene) has been reported recently, and highly purified preparations of this protein have been obtained. We used a nitrocellulose filter assay for measuring the binding of GDP and GTP to the purified protein. Previously p21 antibodies had been used to precipitate p21-guanosine nucleotide complexes from crude extracts containing the protein. Using the filter assay, we find that the v-Ha-ras gene product binds [3H]GDP stoichiometrically. The binding is time-dependent and is faster at 30 degrees C than at 0 degrees C. Optimum binding is obtained in the presence of dithiothreitol and magnesium ions and at pH 7.4. In terms of its GDP binding activity, p21 is heat stable and pronase sensitive. The dissociation constants (Kd) of p21 for [3H]GDP and [3H]GTP, determined by Scatchard analysis, are 6 X 10(-8) M and 2.5 X 10(-8) M, respectively.
Article
A comparison of proteins encoded by normal human ras genes and by mutant rasH or rasK genes activated in human carcinomas revealed no changes in subcellular localization, posttranslational modification, or guanine nucleotide binding associated with activation. Subcellular fractionation indicated that both normal and activated ras proteins were associated exclusively with the membrane fraction. Furthermore, both normal and activated ras proteins exhibited similar degrees of posttranslational acylation. The KD for dGTP binding was 1.0-2.2 X 10(-8) M, with no consistent differences between normal and activated ras proteins. In addition, a survey of 13 possible competing nucleotides revealed no differences in the specificity of nucleotide binding associated with ras gene activation. These results indicate that structural mutations which activate ras gene transforming activity do not alter the protein's known biochemical parameters and in particular do not affect the protein's intrinsic ability to bind guanine nucleotides.
Article
Alteration in gene structure has been shown to occur in some human tumours. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV) and differs from its counterpart in normal cells by a single amino acid. All three of these Ha-ras genes direct the production of similar proteins (p21). While the ras gene appears to be involved in tumour formation in some situations, its role is unclear. The ras protein product (p21) binds guanine nucleotides and has a unique autophosphorylating activity, but no other enzymatic activity has been found. We report here the injection of purified Ha-ras p21 protein, made in Escherichia coli from the gene of BALB-MSV, into NIH 3T3 cells and show that the purified protein itself is sufficient to induce a transformed morphology. In addition, the injected protein stimulates quiescent cells to enter the S-phase of the cell cycle. This result clearly demonstrates that the ras gene functions directly through the protein product. It also establishes an assay for the protein which depends on its activity within a living cell. The transforming activity of a p21 ras protein equivalent to the product of the normal cellular ras gene, is also demonstrated.
  • G L Temeles
  • J B Gibbs
  • J S D 'alonzo
  • I S Sigal
  • E M Scolnick
Temeles, G. L., Gibbs, J. B., D'Alonzo, J. S., Sigal, I. S. & Scolnick, E. M. (1985) Nature (London) 313, 700-703.
  • D Broek
  • N Samiy
  • O Fasano
  • A Fujiyama
  • F Tamanoi
  • J Northrup
  • M Wigler
Broek, D., Samiy, N., Fasano, O., Fujiyama, A., Tamanoi, F., Northrup, J. & Wigler, M. (1985) Cell 41, 763-769.
  • J R Feramisco
  • M Gross
  • T Kamata
  • M Rosenberg
  • R W Sweet
Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M. & Sweet, R. W. (1984) Cell 38, 109-117.
  • J B Gibbs
  • I S Sigal
  • E M Scolnick
Gibbs, J. B., Sigal, I. S. & Scolnick, E. M. (1985) Trends Biochem. Sci. 10, 350-353.
  • T Kataoka
  • S Powers
  • S Cameron
  • O Fasano
  • M Goldfarb
  • J Broach
  • M Wigler
Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J. & Wigler, M. (1985) Cell 40, 19-26.