Analysis of guanine nucleotide bound to ras protein in PC12 cells

The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21(Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTP gamma S [(1987) Mol. Cell. Biol. 7, 4553-4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21.GTP induces the morphological differentiation of PC12 cells.     

Biochemical and biological activity of phosphorylated and non-phosphorylated ras p21 mutants.

In contrast to all cellular ras oncogenes which carry a single activating mutation at codon 12, 13 or 61, all known retroviral ras oncogenes have two mutations at codons 12 and 59. To understand the role of the mutation at codon 59, we have constructed plasmids containing genes for Harvey ras: p21(Gly-12,Thr-59) and p21(Val-12,Thr-59). Escherichia coli expressed proteins and their respective phosphorylated (Pi) and non-phosphorylated (non-Pi) proteins were purified to 95% homogeneity by ion-exchange chromatography and gel filtration. GTPase, autophosphorylation and nucleotide exchange activities of the mutants were studied. When the mutants were microinjected into Xenopus oocytes, the non-phosphorylated forms of p21(Gly-12,Thr-59) and p21(Val-12,Thr-59) showed high activity. Surprisingly, their phosphorylated forms were inactive. These results suggest that threonine at position 59 endows the protein with transforming activity but that phosphorylation of the residue inhibits biological activity. A structural interpretation of the observation is presented.     

Biochemical and biological properties of the human N-ras p21 protein.

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.     

High-level expression of c-H-ras1 fails to fully transform rat-1 cells.

Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.     

Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C.

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.     

Comparison of the conformation and GTP hydrolysing ability of N-terminal ras p21 protein segments

Conformational, GTP binding, and GTP hydrolytic studies are carried out with synthetically prepared N-terminal 34 residue segments (residues 2-35) of p21 ras oncogenic (12-Val) and non-oncogenic (12-Gly) proteins. It was found that these N-terminal regions bind nucleotides through their phosphate groups, and that substitution of valine for glycine produces a more pronounced alpha-helical structure and decreases the conformational flexibility. The glycine containing peptide, when compared to the valine containing analog, catalyses the hydrolysis of GTP 6 times more efficiently. Results suggest that restriction of conformational adaptation may contribute to the transforming capacity of the Val-12 p21 protein.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli

The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.     

Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

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.     

RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

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.     

Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.

In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction. GTP gamma S alone did not induce the contraction in the absence of Ca2+. This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily. The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN. smg p21B, another small GTP-binding protein, was inactive. EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle. The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction. smg p21B was inactive. These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.     

甲胎蛋白对HeLa细胞N-ras、p53和p21~(ras)表达的促进作用

大量研究已证明甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞的增殖具有调节作用 .为探讨AFP对细胞生长促进作用的分子机理 ,采用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,用Northern印迹分析法分析不同作用时间时细胞N rasmRNA的表达以及用Western印迹分析法分析p5 3、p2 1ras的表达 .结果发现 ,在AFP(2 0mg L)作用后 ,HeLa细胞的N rasmRNA、p5 3蛋白质和p2 1ras蛋白质的表达量与对照组比较在 12h和 2 4h时都有明显增加 .AFP的作用均可被抗AFP单克隆抗体所拮抗 .实验结果提示 ,AFP对细胞生长的调节作用可能通过促进这些原癌基因的表达来实现 .     

Activation of p21ras by transforming growth factor beta in epithelial cells.

The transforming growth factor beta (TGF beta) family members are ubiquitously expressed and control a variety of cellular processes by interacting with at least two types of high affinity cell surface receptors. However, the primary signal transduction mechanism of the receptors is unknown. The ras-encoded 21-kDa GTP binding proteins have recently been shown to mediate the effects of other polypeptide growth factors. Here we show that both TGF beta 1 and TGF beta 2 (5 ng/ml) result in a rapid (within 6 or 12 min, respectively) stimulation of GTP bound to p21ras in TGF beta-sensitive intestinal epithelial cells. Further, the CCL64 epithelial cell line, extremely sensitive to growth inhibition by TGF beta, displayed a concentration-dependent increase in GTP bound to p21ras by TGF beta 1 and a rapid activation of p21ras by TGF beta 2. The results provide the first direct evidence for rapid activation of a receptor coupling component for TGF beta in epithelial cells.     

Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.     

Characterization of nucleosidediphosphate (NDP)-kinase-associated GTP binding proteins from human recombinant interleukin 2 (rIL-2)-treated mouse NK cells

Nucleosidediphosphate (NDP)-kinase-associated proteins from rIL-2-treated mouse NK cells have been biochemically characterized. The associated proteins could be separated from partially purified NDP-kinases by the 5-25% glycerol density gradient centrifugation method after treatment with 6 M urea in the presence of 1 mM EDTA. The associated proteins (approx. Mr 20,000) were defined as GTP binding proteins, since only [alpha-32P]GTP was bound to these proteins in the presence of 5 mM Mg2+ at 37 degrees C. We also found that these GTP binding proteins hydrolyzed only GTP in the presence of 5 mM Mg2+. The data presented here for: GTP specific binding activity; GTPase activity; and molecular size (approx. Mr 20,000) of the NDP-kinase-associated GTP binding proteins are similar to those reported for ras oncogene products (p21 proteins).     

The effect of Mg2+ on the guanine nucleotide exchange rate of p21N-ras

There is growing evidence that the protein products of the ras gene family, p21ras, can couple growth factor receptors to intracellular second messenger production and in particular to phosphoinositol lipid turnover. So far, however, there has been no direct proof that the ras proteins function as typical regulatory G proteins. We show here that the human p21N-ras protein, isolated from an Escherichia coli expression system, can exist as a stable GDP complex which exchanges very slowly with exogenous GTP, the half-life of the p21N-ras X GDP complex being around 20 min. However, in low Mg2+ (0.5 microM) the exchange rate is dramatically increased and the half-life of the p21N-ras X GDP complex is less than 30 s. Furthermore, in low Mg2+, the relative binding affinity of the protein for GTP as compared to GDP is increased 10-fold. The effect of low Mg2+ on the exchange rate of both normal and oncogenic mutant p21ras molecules is identical. We propose that removal of Mg2+ in vitro induces a similar conformational change to stimulation in vivo. The properties described here are consistent with a G protein-like activity for p21N-ras.     

Oligomeric structure of p21 ras proteins as determined by radiation inactivation

Using radiation inactivation we determined that p21 ras proteins exhibit an oligomeric target size when assayed both structurally and functionally. Similar target sizes of p21 in ras-transformed cells and in purified preparations of the protein suggested that its structure is homo-oligomeric. p21 monomers were destroyed by radiation with the same target size as the GTP binding activity, indicating the occurrence of a tight association allowing energy transfer between the monomers. Irradiation in the presence of GTP, dithiothreitol, or EDTA did not change the target size. Normal (Gly12) and transforming (Lys12) forms of the protein exhibited similar target sizes. The homo-oligomeric structure suggests that p21 ras proteins do not conform to the structure of monomeric alpha subunits in classical G proteins (alpha beta gamma heterotrimers) and establishes similarities with other homo-oligomeric proteins (such as Escherichia coli CRP) which acquire the active conformation through subunit reorientation upon nucleotide binding.     

An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.     

Pravastatin, a HMG-CoA reductase inhibitor, blocks the cell cycle progression but not Ca2+ influx induced by IGF-I in FRTL-5 cells.

IGF-I, when added to the TSH-primed FRTL-5 cells, induces a long lasting Ca2+ influx, and then, DNA synthesis. Moreover, Ca2+ channel agonist, B AY K8644 can mimic these effects on cell proliferation. We studied the effect of HMG-CoA reductase inhibitor, Pravastatin on IGF-I-induced cell cycle progression in FRTL-5 cells. Pravastatin inhibited DNA synthesis induced both by IGF-I and by BAY K8644. In contrast, Ca2+ influx stimulated by IGF-I was unaffected. These data demonstrate that the signal transduction pathway evoked by IGF-I may possibly involve pravastatin-sensitive process at the downstream step of Ca2+ entry. HMG-CoA reductase inhibitors are known to modulate some cellular signal transduction systems by blocking the membrane attachment of low molecular weight GTP binding proteins such as p21ras. Therefore, pravastatin-sensitive process that we have shown here might possibly involve some of such small G protein.     

Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins

Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP.