Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.     

Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.     

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.     

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Antisense inhibition of ras p21 expression that is sensitive to a point mutation

Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human Ha-ras p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions

Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action.     

Revisiting the structural flexibility of the complex p21(ras)-GTP: the catalytic conformation of the molecular switch II.

The hydrolysis of GTP in p21(ras) triggers conformational changes that regulate the ras/ERK signaling pathway. An important active site residue is Gln61, which has been found to be mutated in 30% of human tumors. The dynamics of the active site conformation is studied by using molecular dynamics simulation of two independent structures of the GTP-bound uncomplexed enzyme. Two distinct conformations of the enzyme are observed, in which the side-chain residue Gln61 is in different orientations. Essential dynamics analysis is used to describe the essential motions in the transition between the two conformations. Results are compared with earlier simulations of p21(ras) and its complex with GTPase activating protein p21-GAP.     

Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.     

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.     

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.     

Regulatory mechanisms for ras proteins.

The proteins encoded by the ras proto-oncogenes play critical roles in normal cellular growth, differentiation and development in addition to their potential for malignant transformation. Several proteins that are involved in the control of the activity of p21ras have now been characterised. p120GAP stimulates the GTPase activity of p21ras and hence acts as a negative regulator of ras proteins. It may be controlled by tyrosine phosphorylation or association with tyrosine phosphorylated proteins. The neurofibromatosis type 1 (NF 1) gene also encodes a potential GTPase activating protein which is likely to be subject to a different control mechanism. Guanosine nucleotide exchange factors for p21ras have now been identified: these may be positive regulators of ras protein function. It appears that p21ras is subject to rapid regulation by several distinct mechanisms which are likely to vary in different cell types; the ras proteins are thereby able to act as very sensitive cellular monitors of the extracellular environment.     

Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C

Stable expression of high levels of activated forms of Haras (T24) or v-Ki-ras by transfection of Chinese hamster lung fibroblasts (CCL39) yielded cells highly tumorigenic in nude mice. Two classes of transformed cells were distinguished, one with moderate p21 expression (10-fold increased) had retained growth factor dependency, the second with higher level of p21 (greater than 50-fold) appeared autonomous for growth. Neither class of transformants expressing Ki-ras or Ha-ras displayed a significant basal activity of polyphosphoinositide-specific phospholipase C, measured either in serum-starved cells or during exponential growth in the presence of growth factors of the tyrosine kinase family (EGF, FGF, insulin). In the growth-factor-dependent class of T24-Ha-ras-transfected cells (clone 39THaB), phospholipase C could be stimulated normally by serum, thrombin and AlF-4. In the more growth autonomous class (clones 39THaC and 39Ki9), release of inositol phosphates after stimulation with thrombin or serum was drastically reduced. This desensitization, apparently at the receptor level since the response to AlF-4 persisted, is, however, not specific to ras expression. We observed it to the same degree in polyoma virus-transformed CCL39 cells. Finally, expression of mutated forms of p21 ras did not abrogate the sensitivity of phospholipase C activation to pertussis toxin. We conclude that the transforming potential of activated forms of p21ras does not result from persistent activation of phospholipase C and that ras GTP-binding proteins cannot substitute for Gp.     

A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange

Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.     

Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.     

NGF-induced neurite regeneration is mediated by a ras-independent pathway in PC12 cells.

A rat pheochromocytoma (PC12) cell line (designated MMTV-M17-5) expressing a dominant inhibitory mutant Ha-ras (Ha-ras Asn 17) protein was used to study nerve growth factor (NGF) induced neurite regeneration. Expression of the mutant p21 completely blocked NGF stimulated process formation in these cells. In contrast, neurite outgrowth induced by NGF treatment of primed MMTV-M17-5 cells was not significantly affected by the presence of Ha-ras Asn 17 protein. These observations suggest that, while ras function is required for NGF induced neuronal differentiation of PC12 cells, it is not needed to mediate NGF stimulated neurite regeneration.