SEARCH · PubMed Health

Results for “RAS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources

PubMed1990

Adhesion of Kirsten-ras+ tumor-progressing and Kirsten-ras- revertant 3T3 cells on fibronectin proteolytic fragments.

Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.

Actins↗

PubMed1995

[Inhibition of ras-dependent transformation by using dominant negative ras mutant N116Y].

Ras p21s are known as molecular switch for signal transduction pathways. They act as intracellular signal transducers of extracellular signals for growth and differentiation. Ras activities are regulated by the rotation between active GTP-bound form and inactive GDP-bound form. This cycle is regulated by the GDP/GTP exchange reaction and intrinsic GTPase activity of ras p21. The N116Y, v-H-ras mutant substituted the asparagine-116 with tyrosine, has dominant negative activity toward normal ras p21, and suppresses ras dependent transformed phenotypes. To investigate the effects of N116Y on ras-mediated signals for transformation, I constructed an inducible vector by recombination of the N116Y mutant to the downstream of human metallothionein promoter, and transfected it into an NIH3T3 cell line transformed by LTR linked normal c-H-ras, 18A. I isolated two 18A cell clones T1 and T6. Both of the cell lines were able to induce the N116Y mutant after the heavy metal treatment. These clones displayed flat reversion within 24 hours. In addition, these clones also inhibited colony formation in soft agar by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or serum stimulation. The N116Y mutant blocked GDP/GTP exchange reaction by each growth stimulation. On the other hand, this mutant could not have sufficient influence upon extracellular signal-regulated kinase 2 (ERK2) phosphorylation, which located downstream of ras-mediated signal transduction, provoked by PDGF and serum stimulation. These results suggest that ERK2 activation is not necessary and sufficient for ras-dependent transformation. There could be a divergency in signal transduction between cell growth and transformation. The signal suppressed by the N116Y mutant may play an important role in cellular transformation.

Animals↗

PubMed1994

Isolation of two novel ras genes in Dictyostelium discoideum; evidence for a complex, developmentally regulated ras gene subfamily.

In Dictyostelium discoideum, three ras genes (rasD, rasG and rasB) and one ras-related gene (rap1) have been previously isolated and characterized, and the deduced amino acid sequence of their predicted protein products share at least 50% sequence identity with the human H-Ras protein. We have now cloned and characterized two additional members of the ras gene subfamily in Dictyostelium, rasC and rasS. These genes are developmentally regulated and unlike the previously isolated Dictyostelium ras genes, maximum levels of their transcripts were detected during aggregation, suggesting that the encoded proteins have distinct functions during aggregation. The rasC cDNA encodes a 189 amino acid protein that is 65% identical to the Dictyostelium RasD and RasG proteins and 56% identical to the human H-Ras protein. The predicted 194 amino acid gene product encoded by rasS is 60% identical to the Dictyostelium RasD and RasG proteins and 54% identical to the human H-Ras protein. Whereas RasD, RasG, RasB and Rap1 are totally conserved in their putative effector domains relative to H-Ras, RasC and RasS have single amino acid substitutions in their effector domains, consistent with the idea that they have unique functions. In RasC, aspartic acid-38 has been replaced by asparagine (D38N), and in RasS, isoleucine-36 has been replaced by leucine (I36L). In addition, both proteins have several differences in the effector-proximal domain, a domain which is believed to play a role in Ras target activation. In RasC, there is a single conservative amino acid change in the canonical sequence of the binding site for the Ras-specific monoclonal antibody Y13-259, and consequently, RasC is less immunoreactive with the antibody than either of the Dictyostelium RasD or RasG proteins. In contrast, RasS, which has three substitutions in the Y13-259 binding site, does not react with the Y13-259 antibody.

Amino Acid Sequence↗

PubMed2004

Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB.

p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons.

Animals↗

PubMed2001

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

3T3 Cells↗

PubMed1996

Stimulation of ras GTPase activity by an anti-ras monoclonal antibody.

Wild-type ras has GTPase activity, and this activity is accelerated substantially by GTPase-activating proteins (GAPs). Oncogenic ras species have an abnormally low intrinsic GTPase activity, and this activity is insensitive to GAPs. We confirmed that the anti-ras monoclonal antibody Y13-238 inhibited GAP activity in vitro, but we also noted that this antibody had GAP activity of its own. We studied the GAP activity of Y13-238 in circumstances in which ras GTPase activity was influenced by the GTPase-inhibitory antibody Y13-259 or by substitutions in ras. The GTPase-inhibitory antibody Y13-259 blocked the GAP associated with Y13-238. A ras species with a substitution in the effector loop that blocked conventional GAP activity was sensitive to stimulation by Y13-238. Both Y13-238 and Y13-259 stimulated the autophosphorylation of Ala59Thr ras. We interpreted these data in terms of a model in which the extrinsic factors influence the ras GTPase reaction by affecting the balance between "committed" and "uncommitted" states. We suggest that there is a mechanism distinct from that exploited by conventional GAPs for stimulating ras GTPase activity.

Amino Acid Sequence↗

PubMed2000

Calcium and calmodulin are essential for Ras-GRF1-mediated activation of the Ras pathway by lysophosphatidic acid.

The exchange factor Ras-GRF1, also called CDC25Mm, couples calcium signaling and G-protein-coupled receptors to Ras and downstream effectors. Here we show that when expressed in different cell lines Ras-GRF1 strongly enhances the level of active Ras (Ras-GTP) and the activity of mitogen-activated protein kinases (MAPK). Moreover, in NIH 3T3 fibroblasts it potentiates the effect of lysophosphatidic acid (LPA) on Ras protein and MAPK activity. Calmodulin and cytosolic free calcium are essential for Ras and MAPK activation induced by LPA and mediated by Ras-GRF1, as shown by the finding that BAPTA-AM, an intracellular calcium chelator, and calmodulin inhibitors completely abolished this effect. This report demonstrates the relevance of calmodulin in addition to calcium for the response of Ras-GRF1 to LPA.

3T3 Cells↗

PubMed1995

rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21.

We have recently found that ralGDS family members (RGL and ralGDS) are putative effector proteins of ras p21. rap1 p21 is a small GTP-binding protein which has the same amino acid sequence as the effector loop of ras p21. We examined the effect of rap1 p21 on the interaction of ras p21 with RGL. The GTP-bound form of rap1 p21 interacted with RGL as well as did ras p21. rap1 p21 inhibited the interaction of ras p21 with RGL. RGL was phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). Phosphorylation of RGL did not affect its binding to ras p21 and rap1 p21 under the conditions that phosphorylation of Raf-1 reduced its affinity for ras p21. These results demonstrate that rap1 p21 but not protein kinase A regulates the interaction of ras p21 with RGL and suggest that rap1 p21 and protein kinase A may cooperate to distinguish the signal or ras p21 to RGL from that to Raf-1.

Cloning, Molecular↗

PubMed1992

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.

Adenylyl Cyclases↗

PubMed2002

ras Gene mutations and expression of Ras signal transduction mediators in gastric adenocarcinomas.

OBJECTIVE: To investigate ras gene alteration in human gastric adenocarcinomas and its potential relationship to ras signal transduction mediators. DESIGN: Genomic DNA from 104 gastric tumors were analyzed by sequencing of polymerase chain reaction-amplified products for the presence of ras mutations. All the samples were further investigated with the use of immunohistochemical analysis for ERK1 and ERK2. SETTING: Tertiary care teaching hospital. PATIENTS: Seventy patients from a Korean population and 34 from a Midwestern US population composed of white Americans and African Americans. RESULTS: Fifteen tumors (14%) were positive for either H-ras or K-ras mutation: 9 (13%) of 70 Korean patients and 6 (18%) of 34 US patients. Seven (78%) of the 9 mutated tumors from Korean patients and all 6 (100%) from the US patients were intestinal-type lesions. Either ERK1 and/or ERK2 was overexpressed in 68 samples (65%). No association was established between ras mutations and overexpression of ERK1/2. However, the correlation between ERK1/2 and progression (early vs late) was statistically significant (P =.007). CONCLUSIONS: These data suggest that ras mutations are uncommon in gastric adenocarcinomas and that differing racial and/or geographic mechanisms may not underlie ras gene alteration. Most ras mutations were, however, observed in the group of intestinal-type samples, supporting the different genetic mechanisms of carcinogenesis between the intestinal- and diffuse-type tumors. It is noteworthy that enhanced ERK1/2 activity could be one of the characteristics of tumor invasiveness in gastric cancers.

Adenocarcinoma↗

PubMed1992

Altered H1 histamine receptor signaling in Balb/3T3 cells transformed by v-K-ras and v-H-ras oncogenes.

Transformation of Balb/3T3 cells with the v-K-ras oncogene resulted in the expression of functional Ca(2+)-mobilizing receptors for histamine, whereas v-H-ras-transformed Balb/3T3 cells failed to show a similar response to histamine. Stimulation of histamine receptors in v-K-ras-transformed cells produced a dose-dependent increase in intracellular free calcium ([Ca2+]i), which was inhibited by the H1 histamine antagonist pyrilamine but unaffected by the H2 histamine receptor antagonist cimetidine. Histamine-mediated elevation of [Ca2+]i was partially inhibited by the removal of extracellular Ca2+, which indicates that the H1 histamine receptors mobilize intracellular Ca2+ and also promote Ca2+ influx. H1 histamine receptors were identified in both v-K-ras- and v-H-ras-transformed Balb/3T3 cells, but not in untransformed cells, using the specific H1 antagonist [3H]-pyrilamine. Transformation of Balb/3T3 cells with the viral ras oncogene results in a complex regulation of H1 histamine receptors. K-ras and H-ras transformation results in the expression of H1 histamine receptors; however, H1 receptor expression and Ca2+ mobilization are uncoupled in v-H-ras-transformed cells.

3T3 Cells↗

PubMed1993

Kinetics of interaction between normal and proline 12 Ras and the GTPase-activating proteins, p120-GAP and neurofibromin. The significance of the intrinsic GTPase rate in determining the transforming ability of ras.

Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.

Cell Transformation, Neoplastic↗

PubMed1995

Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung.

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.

Adenocarcinoma↗

PubMed1996

Identification of a potential physiological substrate for oncogenic Ras-activated protein kinases in activated Xenopus egg extracts: correlation with oncogenic Ras-induced cell cycle arrest.

Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases.

14-3-3 Proteins↗

PubMed1995

Purification of histidine-tagged ras and its use in the detection of ras binding proteins.

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts of E. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni(2+)-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litre E. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search for ras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidate ras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identify ras binding proteins present in cellular extracts.

Animals↗

PubMed2001

The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors.

Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.

GTPase-Activating Proteins↗

PubMed2002

Isoprenoids influence expression of Ras and Ras-related proteins.

Mevalonate depletion by inhibition of hydroxymethylglutaryl coenzyme A reductase impairs post-translational processing of Ras and Ras-related proteins. We have previously shown that this mevalonate depletion also leads to the upregulation of Ras, Rap1a, RhoA, and RhoB. This upregulation may result from global inhibition of isoprenylation or depletion of key regulatory isoprenoid species. Studies utilizing specific isoprenoid pyrophosphates in mevalonate-depleted cells reveal that farnesyl pyrophosphate (FPP) restores Ras processing and prevents RhoB upregulation while geranylgeranyl pyrophosphate (GGPP) restores Rap1a processing and prevents RhoA and RhoB upregulation. Either FPP or GGPP completely prevents lovastatin-induced upregulation of RhoB mRNA. Inhibition of FPP or squalene synthase allowed for the further identification of the putative regulatory species. Studies involving the specific isoprenyl transferase inhibitors FTI-277 and GGTI-286 demonstrate that selective inhibition of protein isoprenylation does not mimic lovastatin's ability to increase Ras and RhoA synthesis, decrease Ras and RhoA degradation, increase RhoB mRNA, or increase total levels of Ras, Rap1a, RhoA, and RhoB. In aggregate, these findings reveal a novel role and mechanism for isoprenoids to influence levels of Ras and Ras-related proteins.

Antineoplastic Agents↗

PubMed2004

Signal processing at the Ras circuit: what shapes Ras activation patterns?

A systems biology approach is applied to gain a quantitative understanding of the integration of signalling by the small GTPase Ras. The Ras protein acts as a critical switch in response to signals that determine the cell's fate. In unstimulated cells, Ras switching between an inactive GDP-binding and active GTP-binding state is controlled by the intrinsic catalytic activities of Ras. The calculated high sensitivity of the basal Ras-GTP fraction to changes in the rate constant of GTP-hydrolysis by Ras can account for the carcinogenic potential of Ras mutants with decreased GTPase activities. Extracelluar stimuli initiate Ras interactions with GDP/GTP exchange factors such as SOS, and GTP-hydrolysis activating proteins such as RasGAP. Our data on freshly isolated hepatocytes stimulated with epidermal growth factor (EGF) show transient SOS activation and sustained Ras-GTP patterns. We demonstrate that these dose-response data can only be explained by transient RasGAP activitation, and not by merely switching off the SOS signal, e.g. by inhibitory phosphorylation of SOS. A transient RasGAP activity can be brought about by a number of mechanisms. A comprehensive kinetic model of the EGF receptor (EGFR) network was developed to explore feasible molecular scenarios, including the receptor-mediated recruitment of SOS and RasGAP to the plasma membrane, phosphorylation of RasGAP and p190 RhoGAP by soluble tyrosine kinases, and RasGAP interactions with phosphoinositides and p190 RhoGAP. We show that a transient RasGAP association with EGFR followed by the capture of RasGAP through the formation of complexes with p190 RhoGAP can account for data on hepatocytes. In summary, our results demonstrate that a combination of experimental monitoring and integrated dynamic analysis is capable of dissecting regulatory mechanisms that govern cellular signal transduction.

Animals↗