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Jacqueline M Bentel - One of the best experts on this subject based on the ideXlab platform.
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are all Ras mutations the same coexisting kRas and nRas mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma
Journal of Clinical Pathology, 2015Co-Authors: Nina Vagaja, Jeremy Parry, Dugald Mccallum, M A Thomas, Jacqueline M BentelAbstract:Mutations of the human Kirsten rat sarcoma viral oncogene homologue ( KRas ) and the highly homologous human neuroblastoma Ras viral oncogene homologue ( NRas ) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRas c.35G>T (G12V) and NRas c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRas and NRas mutations, supporting their common origin. While KRas mutations are common in colorectal cancers, NRas mutations are relatively rare and the coexistence of multiple Ras mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of Ras. Recent experimental evidence has suggested that KRas and NRas may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of Ras family members and mutant forms of Ras in the colon form important considerations for the development of Ras-targeting therapeutics.
Michael F. Moran - One of the best experts on this subject based on the ideXlab platform.
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Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2
Molecular and Cellular Biology, 2001Co-Authors: Carmen L. De Hoog, Jackie A. Koehler, Marni D. Goldstein, Paul J. Taylor, Daniel Figeys, Michael F. MoranAbstract:Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.
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Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.
Molecular and Cellular Biology, 1997Co-Authors: Zhixiang Wang, L J Zhang, H Chen, Michael F. MoranAbstract:Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.
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A murine CDC25/Ras-GRF-related protein implicated in Ras regulation.
Developmental Genetics, 1993Co-Authors: Luping Chen, Li‐jia Zhang, Peter A. Greer, Pierre S. Tung, Michael F. MoranAbstract:A partial cDNA encoding a novel putative p2, Ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/Ras-GRF, but is more similar to yeast CDC25 than to other Ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/Ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/Ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate Ras-dependent proliferation and differentiation in early mouse development. © 1993Wiley-Liss, Inc.
Mark R. Philips - One of the best experts on this subject based on the ideXlab platform.
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targeting Ras membrane association back to the future for anti Ras drug discovery
Clinical Cancer Research, 2015Co-Authors: Mark R. PhilipsAbstract:Ras proteins require membrane association for their biologic activity, making this association a logical target for anti-Ras therapeutics. Lipid modification of Ras proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransfeRase inhibitors (FTI) were developed as potential anti-Ras drugs. The lack of efficacy of FTIs as anticancer drugs was widely seen as indicating that blocking Ras membrane association was a flawed approach to cancer treatment. However, a deeper understanding of Ras modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRas and NRas, which are the Ras isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking Ras membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding Ras isoform trafficking and the regulation of Ras subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates Ras interaction with the plasma membrane, endomembranes, and cytosol, and of the potential importance of Ras chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated posttranslational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting Ras membrane association and subcellular localization for anti-Ras drug development. Clin Cancer Res; 21(8); 1819–27. ©2015 AACR . See all articles in this CCR Focus section, “Targeting Ras-Driven Cancers.”
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Compartmentalized signalling of Ras
Biochemical Society Transactions, 2005Co-Authors: Mark R. PhilipsAbstract:Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCγ (phospholipase Cγ)/DAG (diacylglycerol)+Ca 2+ /RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization.
Allan Balmain - One of the best experts on this subject based on the ideXlab platform.
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interactions between wild type and mutant Ras genes in lung and skin carcinogenesis
Oncogene, 2013Co-Authors: Minh D To, Reyno Del Rosario, Peter M K Westcott, Karl Luke Banta, Allan BalmainAbstract:Ras oncogenes (HRas, KRas and NRas) are important drivers of carcinogenesis. However, tumors with Ras mutations often show loss of the corresponding wild-type (WT) allele, suggesting that proto-oncogenic forms of Ras can function as a suppressor of carcinogenesis. In vitro studies also suggest that WT Ras proteins can suppress the tumorigenic properties of alternate mutant Ras family members, but in vivo evidence for these heterologous interactions is lacking. We have investigated the genetic interactions between different combinations of mutant and WT Ras alleles in vivo using carcinogen-induced lung and skin carcinogenesis in mice with targeted deletion of different Ras family members. The major suppressor effect of WT KRas is observed only in mutant KRas-driven lung carcinogenesis, where loss of one KRas allele led to increased tumor number and size. Deletion of one HRas allele dramatically reduced the number of skin papillomas with HRas mutations, consistent with HRas as the major target of mutation in these tumors. However, skin carcinoma numbers were very similar, suggesting that WT HRas functions as a suppressor of progression from papillomas to invasive squamous carcinomas. In the skin, the KRas proto-oncogene functions cooperatively with mutant HRas to promote papilloma development, although the effect is relatively small. In contRast, the HRas proto-oncogene attenuated the activity of mutant KRas in lung carcinogenesis. Interestingly, loss of NRas increased the number of mutant KRas-induced lung tumors, but decreased the number of mutant HRas-induced skin papillomas. These results show that the strongest suppressor effects of WT Ras are only seen in the context of mutation of the cognate Ras protein, and only relatively weak effects are detected on tumor development induced by mutations in alternative family members. The data also underscore the complex and context-dependent nature of interactions between proto-oncogenic and oncogenic forms of different Ras family members during tumor development.
Nina Vagaja - One of the best experts on this subject based on the ideXlab platform.
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are all Ras mutations the same coexisting kRas and nRas mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma
Journal of Clinical Pathology, 2015Co-Authors: Nina Vagaja, Jeremy Parry, Dugald Mccallum, M A Thomas, Jacqueline M BentelAbstract:Mutations of the human Kirsten rat sarcoma viral oncogene homologue ( KRas ) and the highly homologous human neuroblastoma Ras viral oncogene homologue ( NRas ) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRas c.35G>T (G12V) and NRas c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRas and NRas mutations, supporting their common origin. While KRas mutations are common in colorectal cancers, NRas mutations are relatively rare and the coexistence of multiple Ras mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of Ras. Recent experimental evidence has suggested that KRas and NRas may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of Ras family members and mutant forms of Ras in the colon form important considerations for the development of Ras-targeting therapeutics.
