Journal articles on the topic 'Ras proteins'
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1
Takács, Tamás, Gyöngyi Kudlik, Anita Kurilla, Bálint Szeder, László Buday, and Virag Vas. "The effects of mutant Ras proteins on the cell signalome." Cancer and Metastasis Reviews 39, no. 4 (July 9, 2020): 1051–65. http://dx.doi.org/10.1007/s10555-020-09912-8.
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AbstractThe genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells.
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Jelinek, T., P. Dent, T. W. Sturgill, and M. J. Weber. "Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation." Molecular and Cellular Biology 16, no. 3 (March 1996): 1027–34. http://dx.doi.org/10.1128/mcb.16.3.1027.
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Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.
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Bhattacharya, M., A. V. Babwah, and S. S. G. Ferguson. "Small GTP-binding protein-coupled receptors." Biochemical Society Transactions 32, no. 6 (October 26, 2004): 1040–44. http://dx.doi.org/10.1042/bst0321040.
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Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20–30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.
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Tang, Yi, Jong Yu, and Jeffrey Field. "Signals from the Ras, Rac, and Rho GTPases Converge on the Pak Protein Kinase in Rat-1 Fibroblasts." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1881–91. http://dx.doi.org/10.1128/mcb.19.3.1881.
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ABSTRACT Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65Pak). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.
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Goi, Takanori, Gabriel Rusanescu, Takeshi Urano, and Larry A. Feig. "Ral-Specific Guanine Nucleotide Exchange Factor Activity Opposes Other Ras Effectors in PC12 Cells by Inhibiting Neurite Outgrowth." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1731–41. http://dx.doi.org/10.1128/mcb.19.3.1731.
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ABSTRACT Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only ∼20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.
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Reddy, E. Premkumar, Sai Krishna Divakar, Rodrigo Vasquez-Del Carpio, Kaushik Dutta, Stacey J. Baker, Ramana Reddy, and Aneel K. Aggarwal. "Rigosertib Blocks RAS Signaling By Acting As a Small Molecule RAS Mimetic That Binds to the RAS-Binding Domains of RAS Effector Proteins." Blood 124, no. 21 (December 6, 2014): 5616. http://dx.doi.org/10.1182/blood.v124.21.5616.5616.
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Abstract Oncogenic activation of RAS via point mutations occurs in more than 30% of all human cancers, including hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Investigations to understand the critical biochemical and biological mechanisms of RAS function are at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play an essential role in oncogenic RAS function, inhibiting them constitutes an attractive and important therapeutic approach for myeloid neoplasias and other cancers. Rigosertib is a novel styryl benzyl sulfone, which is in a Phase III clinical trial (ONTIME) for MDS. Here, we delineate the way rigosertib interacts with the RBDs of several RAS effector proteins: RAF, the PI3K family of proteins and RalGDS. To identify residues in the B-RAF RBD that interact with rigosertib, we recorded a series of 15N-1H HSQC spectra of 15N-labeled B-RAF RBD with increasing concentration of rigosertib. Strikingly, the chemical shift perturbations caused by addition of rigosertib are localized to the very region of the B-RAF-RBD implicated in RAS binding, namely the beta1 and beta2 strands and alpha3 helix (Fig 1). Additionally, this cluster of residues with largest chemical shift perturbation contains many of the same residues involved in RAS binding, namely Ile156, Lys164, Arg166, Thr167, Val168, Ala184 and Met187. These key residues are conserved within RAF RBDs, suggesting that rigosertib would bind to similar regions of the A- and c-RAF RBDs. Next, we examined the binding of rigosertib and GTP-RAS to wild type and mutant forms of c-RAF RBD that harbor mutations in residues that mediate binding to rigosertib. Our studies show that all mutations that cause dissociation of GTP-RAS binding also inhibit rigosertib binding to these mutant proteins. Taken together, the chemical shift data and mutagenesis data provide powerful evidence that rigosertib binds the B-RAF RBD at the same location as the RAS switch I region. A consequence of inhibiting RAS binding to RAF appears to be a block in growth factor-induced activation of RAF kinase activity. We also show that a result of this block in RAS/RAF interactions is an inability of RAF proteins to form dimers and activate MEK and ERK. This block in the activation of MEK/ERK pathways can be seen in cells that express wild-type RAS and RAF proteins (HeLa), in cells that express a constitutively active form of oncogenic RAS (HeLa-N-RAS-G12D), and in cells that exhibit amplification of EGF receptors (A431). Rigosertib also inhibits the phosphorylation of c-RAF serine 338, which has been shown to be essential for the activation of its kinase activity and for its association with and activation of PLK-1. Our results showing rigosertib-mediated inhibition of the PLK-1/RAF interaction might help explain the ability of this compound to induce mitotic arrest of human tumor cells and the ability of rigosertib to reduce blast counts in MDS patients (Seetharam et al, Leuk Res 2012). We have also demonstrated the binding of rigosertib to the RBDs of the PI3K family of kinases and RalGDS, both of which constitute important effectors of RAS. A consequence of the interaction of rigosertib with the RBD domains of PI3Ks appears to be a block in growth factor-induced AKT activation. These studies suggest that the disruption of multiple RAS-driven signaling pathways by rigosertib is mediated via rigosertib’s binding to RBDs of RAS effector proteins, leading to their inactivation. Figure 1 Figure 1. Disclosures Reddy: Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.
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Ramocki, M. B., S. E. Johnson, M. A. White, C. L. Ashendel, S. F. Konieczny, and E. J. Taparowsky. "Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis." Molecular and Cellular Biology 17, no. 7 (July 1997): 3547–55. http://dx.doi.org/10.1128/mcb.17.7.3547.
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The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.
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Stang, S., D. Bottorff, and J. C. Stone. "Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells." Molecular and Cellular Biology 17, no. 6 (June 1997): 3047–55. http://dx.doi.org/10.1128/mcb.17.6.3047.
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v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.
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Tang, Y., Z. Chen, D. Ambrose, J. Liu, J. B. Gibbs, J. Chernoff, and J. Field. "Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts." Molecular and Cellular Biology 17, no. 8 (August 1997): 4454–64. http://dx.doi.org/10.1128/mcb.17.8.4454.
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Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.
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KELLEY, Grant G., Sarah E. REKS, and Alan V. SMRCKA. "Hormonal regulation of phospholipase Cepsilon through distinct and overlapping pathways involving G12 and Ras family G-proteins." Biochemical Journal 378, no. 1 (February 15, 2004): 129–39. http://dx.doi.org/10.1042/bj20031370.
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PLC∊ (phospholipase C∊) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLC∊. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLC∊ overexpressed in COS-7 cells. EGF stimulated PLC∊ in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLC∊ by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLC∊ and found that Gα12, Gα13, Rho, Rac and Ral stimulate PLC∊ in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLC∊ in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that Gα12/Gα13 and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLC∊. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLC∊ by distinct and overlapping pathways.
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Hall, Alan. "Ras-related proteins." Current Opinion in Cell Biology 5, no. 2 (April 1993): 265–68. http://dx.doi.org/10.1016/0955-0674(93)90114-6.
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Graham, S. M., A. B. Vojtek, S. Y. Huff, A. D. Cox, G. J. Clark, J. A. Cooper, and C. J. Der. "TC21 causes transformation by Raf-independent signaling pathways." Molecular and Cellular Biology 16, no. 11 (November 1996): 6132–40. http://dx.doi.org/10.1128/mcb.16.11.6132.
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Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.
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Khosravi-Far, R., M. A. White, J. K. Westwick, P. A. Solski, M. Chrzanowska-Wodnicka, L. Van Aelst, M. H. Wigler, and C. J. Der. "Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation." Molecular and Cellular Biology 16, no. 7 (July 1996): 3923–33. http://dx.doi.org/10.1128/mcb.16.7.3923.
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Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.
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Prior, I. A., and J. F. Hancock. "Compartmentalization of Ras proteins." Journal of Cell Science 114, no. 9 (May 1, 2001): 1603–8. http://dx.doi.org/10.1242/jcs.114.9.1603.
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The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effectors and exchange factors.
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Beranger, F., H. Paterson, S. Powers, J. de Gunzburg, and J. F. Hancock. "The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization." Molecular and Cellular Biology 14, no. 1 (January 1994): 744–58. http://dx.doi.org/10.1128/mcb.14.1.744-758.1994.
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C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.
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Beranger, F., H. Paterson, S. Powers, J. de Gunzburg, and J. F. Hancock. "The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization." Molecular and Cellular Biology 14, no. 1 (January 1994): 744–58. http://dx.doi.org/10.1128/mcb.14.1.744.
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C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.
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Marty, Caroline, Darren D. Browning, and Richard D. Ye. "Identification of Tetratricopeptide Repeat 1 as an Adaptor Protein That Interacts with Heterotrimeric G Proteins and the Small GTPase Ras." Molecular and Cellular Biology 23, no. 11 (June 1, 2003): 3847–58. http://dx.doi.org/10.1128/mcb.23.11.3847-3858.2003.
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ABSTRACT The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Gα16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Gα proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Gα16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Gα16 while maintaining the interaction with Gα16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Gαi-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Gα proteins that may be involved in protein-protein interaction relating to G-protein signaling.
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Cox, A. D., M. M. Hisaka, J. E. Buss, and C. J. Der. "Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein." Molecular and Cellular Biology 12, no. 6 (June 1992): 2606–15. http://dx.doi.org/10.1128/mcb.12.6.2606-2615.1992.
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While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.
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Cox, A. D., M. M. Hisaka, J. E. Buss, and C. J. Der. "Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein." Molecular and Cellular Biology 12, no. 6 (June 1992): 2606–15. http://dx.doi.org/10.1128/mcb.12.6.2606.
Full textAbstract:
While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.
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Simanshu, Dhirendra K. "Abstract IA05: Uncovering new structural insights into RAS interactions with effectors and regulators." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): IA05. http://dx.doi.org/10.1158/1557-3125.ras23-ia05.
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Abstract RAS proteins, which act as binary molecular switches, are implicated in approximately 20% of all cancer cases and are known to play a critical role in the initiation and progression of some of the deadliest cancers, such as lung, colon, and pancreatic cancers. When RAS proteins are in the GTP-bound state, they interact with various effector proteins, such as RAF Kinase, PI 3-Kinase, and RalGDS, leading to the activation of multiple signaling pathways in the cell. Our recent structural studies, including investigations into the KRAS-SIN1 (RBD-PH domain), KRAS-RAF1 (RBD-CRD), and SHOC2-MRAS-PP1C complex, have provided new insights into the vital role played by the switch-II and interswitch regions in the interaction between RAS and downstream effectors and regulators. These findings extend beyond the earlier observations that the switch-I region of RAS proteins alone plays a more significant role in RAS-effector interactions. Furthermore, research into the KRAS4a-SIN1 complex and the reported interaction between KRAS4a and hexokinase have given us a glimpse into the isoform-specific RAS-effector interaction. Finally, the investigation of the SHOC2-MRAS-PP1C complex has also revealed the crucial role played by other members of the RAS subfamily, such as MRAS, in RAF activation by canonical RAS isoforms. Collectively, these studies have shed new light on the mechanisms by which RAS interacts with downstream effectors and regulators. Citation Format: Dhirendra K. Simanshu. Uncovering new structural insights into RAS interactions with effectors and regulators [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA05.
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Dent, P., D. B. Reardon, D. K. Morrison, and T. W. Sturgill. "Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro." Molecular and Cellular Biology 15, no. 8 (August 1995): 4125–35. http://dx.doi.org/10.1128/mcb.15.8.4125.
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The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.
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Tong, L., M. Milburn, A. de Vos, and S. Kim. "Structure of ras proteins." Science 245, no. 4915 (July 21, 1989): 244. http://dx.doi.org/10.1126/science.2665078.
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Chardin, Pierre. "The ras superfamily proteins." Biochimie 70, no. 7 (July 1988): 865–68. http://dx.doi.org/10.1016/0300-9084(88)90226-x.
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Rowinsky, Eric K., Jolene J. Windle, and Daniel D. Von Hoff. "Ras Protein Farnesyltransferase: A Strategic Target for Anticancer Therapeutic Development." Journal of Clinical Oncology 17, no. 11 (November 1999): 3631–52. http://dx.doi.org/10.1200/jco.1999.17.11.3631.
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ABSTRACT: Ras proteins are guanine nucleotide–binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first—and most critical—modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.
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Li, Weiquan, Min Han, and Kun-Liang Guan. "The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf." Genes & Development 14, no. 8 (April 15, 2000): 895–900. http://dx.doi.org/10.1101/gad.14.8.895.
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Caenorhabditis elegans sur-8 encodes a positive regulator of Ras signaling. We investigated the mechanism by which the human Sur-8 homolog can positively regulate Ras–MAP kinase signaling in mammalian cells. Sur-8 expression enhances Ras- or EGF-induced Raf and ERK activation but has no effect on ERK activation induced by active Raf or MEK. Furthermore, Sur-8 expression does not increase AKT or JNK activation. Sur-8 interacts with Ras and Raf and is able to form a ternary complex with the two proteins. Thus, Sur-8 may function as a scaffold that enhances Ras–MAP kinase signal transduction by facilitating the interaction between Ras and Raf.
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Molzan, Manuela, Benjamin Schumacher, Corinna Ottmann, Angela Baljuls, Lisa Polzien, Michael Weyand, Philipp Thiel, et al. "Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling." Molecular and Cellular Biology 30, no. 19 (August 2, 2010): 4698–711. http://dx.doi.org/10.1128/mcb.01636-09.
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ABSTRACT The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser259, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS259 crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer259/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.
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Sealover, Nancy, Bridget Finniff, and Robert Kortum. "Abstract B033: Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): B033. http://dx.doi.org/10.1158/1557-3125.ras23-b033.
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Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS effectively activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy Sealover, Bridget Finniff, Robert Kortum. Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B033.
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Loirand, Gervaise, Vincent Sauzeau, and Pierre Pacaud. "Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects." Physiological Reviews 93, no. 4 (October 2013): 1659–720. http://dx.doi.org/10.1152/physrev.00021.2012.
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Small G proteins exist in eukaryotes from yeast to human and constitute the Ras superfamily comprising more than 100 members. This superfamily is structurally classified into five families: the Ras, Rho, Rab, Arf, and Ran families that control a wide variety of cell and biological functions through highly coordinated regulation processes. Increasing evidence has accumulated to identify small G proteins and their regulators as key players of the cardiovascular physiology that control a large panel of cardiac (heart rhythm, contraction, hypertrophy) and vascular functions (angiogenesis, vascular permeability, vasoconstriction). Indeed, basal Ras protein activity is required for homeostatic functions in physiological conditions, but sustained overactivation of Ras proteins or spatiotemporal dysregulation of Ras signaling pathways has pathological consequences in the cardiovascular system. The primary object of this review is to provide a comprehensive overview of the current progress in our understanding of the role of small G proteins and their regulators in cardiovascular physiology and pathologies.
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Takai, Yoshimi, Takuya Sasaki, and Takashi Matozaki. "Small GTP-Binding Proteins." Physiological Reviews 81, no. 1 (January 1, 2001): 153–208. http://dx.doi.org/10.1152/physrev.2001.81.1.153.
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Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.
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Nagahama, Masahiro, Akiko Ohkubo, Masataka Oda, Keiko Kobayashi, Katsuhiko Amimoto, Kazuaki Miyamoto, and Jun Sakurai. "Clostridium perfringensTpeL Glycosylates the Rac and Ras Subfamily Proteins." Infection and Immunity 79, no. 2 (November 22, 2010): 905–10. http://dx.doi.org/10.1128/iai.01019-10.
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ABSTRACTClostridium perfringensTpeL belongs to a family of large clostridial cytotoxins that encompassesClostridium difficiletoxin A (TcdA) and B (TcdB) andClostridium sordelliilethal toxin (TcsL). We report here the identification of the TpeL-catalyzed modification of small GTPases. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the rounding of Vero cells and the glycosylation of cellular Rac1. Among several hexoses tested, UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) served as cosubstrates for TpeL1-525-catalyzed modifications. TpeL1-525 catalyzed the incorporation of UDP-Glc into Ha-Ras, Rap1B, and RalA and of UDP-GlcNAc into Rac1, Ha-Ras, Rap1B, and RalA. In Rac1, TpeL and TcdB share the same acceptor amino acid for glycosylation, Thr-35. In Vero cells treated with TpeL1-525 in the presence of SLO, glycosylation leads to a translocation of the majority of Rac1 and Ha-Ras to the membrane. We demonstrate for first time that TpeL uses both UDP-GlcNAc and UDP-Glc as donor cosubstrates and modifies the Rac1 and Ras subfamily by glycosylation to mediate its cytotoxic effects.
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Singh, Swati, and Matthew J. Smith. "RAS GTPase signalling to alternative effector pathways." Biochemical Society Transactions 48, no. 5 (October 14, 2020): 2241–52. http://dx.doi.org/10.1042/bst20200506.
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RAS GTPases are fundamental regulators of development and drivers of an extraordinary number of human cancers. RAS oncoproteins constitutively signal through downstream effector proteins, triggering cancer initiation, progression and metastasis. In the absence of targeted therapeutics to mutant RAS itself, inhibitors of downstream pathways controlled by the effector kinases RAF and PI3K have become tools in the treatment of RAS-driven tumours. Unfortunately, the efficacy of this approach has been greatly minimized by the prevalence of acquired drug resistance. Decades of research have established that RAS signalling is highly complex, and in addition to RAF and PI3K these small GTPase proteins can interact with an array of alternative effectors that feature RAS binding domains. The consequence of RAS binding to these effectors remains relatively unexplored, but these pathways may provide targets for combinatorial therapeutics. We discuss here three candidate alternative effectors: RALGEFs, RASSF5 and AFDN, detailing their interaction with RAS GTPases and their biological significance. The metastatic nature of RAS-driven cancers suggests more attention should be granted to these alternate pathways, as they are highly implicated in the regulation of cell adhesion, polarity, cell size and cytoskeletal architecture.
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Day, Gwo-Jen, Raymond D. Mosteller, and Daniel Broek. "Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner." Molecular and Cellular Biology 18, no. 12 (December 1, 1998): 7444–54. http://dx.doi.org/10.1128/mcb.18.12.7444.
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ABSTRACT The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.
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Sandoval, Carolina Ortiz, and Thomas Simmen. "Rab proteins of the endoplasmic reticulum: functions and interactors." Biochemical Society Transactions 40, no. 6 (November 21, 2012): 1426–32. http://dx.doi.org/10.1042/bst20120158.
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Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER–Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.
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Williams, Thomas D., Peggy I. Paschke, and Robert R. Kay. "Function of small GTPases in Dictyostelium macropinocytosis." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1765 (December 17, 2018): 20180150. http://dx.doi.org/10.1098/rstb.2018.0150.
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Macropinocytosis—the large-scale, non-specific uptake of fluid by cells—is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular ruffles around regions of membrane defined by a patch of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the activated forms of the small G-proteins Ras and Rac. When this ruffle closes, a vesicle of the medium is delivered to the cell interior for further processing. It is accepted that PIP3 is required for efficient macropinocytosis. Here, we assess the roles of Ras and Rac in Dictyostelium macropinocytosis. Gain-of-function experiments show that macropinocytosis is stimulated by persistent Ras activation and genetic analysis suggests that RasG and RasS are the key Ras proteins involved. Among the activating guanine exchange factors (GEFs), GefF is implicated in macropinocytosis by an insertional mutant. The individual roles of Rho family proteins are little understood but activation of at least some may be independent of PIP3. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.
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Zondag, Gerben C. M., Eva E. Evers, Jean P. ten Klooster, Lennert Janssen, Rob A. van der Kammen, and John G. Collard. "Oncogenic Ras Downregulates Rac Activity, Which Leads to Increased Rho Activity and Epithelial–Mesenchymal Transition." Journal of Cell Biology 149, no. 4 (May 15, 2000): 775–82. http://dx.doi.org/10.1083/jcb.149.4.775.
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Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial–mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.
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Goldfinger, Lawrence E., Celeste Ptak, Erin D. Jeffery, Jeffrey Shabanowitz, Donald F. Hunt, and Mark H. Ginsberg. "RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration." Journal of Cell Biology 174, no. 6 (September 11, 2006): 877–88. http://dx.doi.org/10.1083/jcb.200603111.
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The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.
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Bolomsky, Arnold, and Ryan M. Young. "Abstract A037: Oncogenic RAS signals from lysosomes to activate mTORC1 in multiple myeloma." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): A037. http://dx.doi.org/10.1158/1557-3125.ras23-a037.
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Abstract Oncogenic mutations of both NRAS and KRAS are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in MM have remained enigmatic, and there is little evidence to support a dominant role for classical MAPK signaling downstream of RAS in this disease. We recently used an unbiased proteogenomic approach to dissect RAS signaling in MM and discover that mutant isoforms of RAS organized a signaling complex with the amino acid transporter, SLC3A2, and MTOR on lysosomes, which directly activated mTORC1 by co-opting amino acid sensing pathways (PMID: 36115844). To gain further insights into this novel mode of RAS signaling located away from the plasma membrane, we have employed mass spectrometry to characterize proteins associated with lysosomes isolated from MM cell lines. We observed that RAS isoforms, SLC3A2 and several components of the mTORC1 signalosome were purified with lysosomes, consistent with RAS activation of mTORC1 on lysosomes. To identify novel regulators of oncogenic RAS signaling at the lysosome, we used phospho-proteomics to identify proteins with significantly reduced levels of phosphorylation following acute inhibition of KRAS activity with the KRAS G12D inhibitor, MRTX1133, in a MM cell line bearing a KRAS G12D mutation. Many of these phospho-proteins were also associated with lysosomes, including MTOR, which had substantially reduced phosphorylation at the amino-acid dependent phosphorylation site S1261 following KRAS inhibition. Pathway analysis of proteins present in lysosomes and phosphorylated downstream of oncogenic KRAS showed enrichment for proteins that regulate intracellular trafficking. Among these proteins, we found that disruption of a RAB GAP essential for survival in RAS-dependent MM cells substantially reduced associations between RAS and MTOR and nearly abolished RAS-dependent mTORC1 activity. These data suggest that mutant RAS utilizes lysosomal resident proteins to coordinate the colocalization of itself with mTORC1 on lysosomes to drive pathogenic signaling in MM. More generally, these studies suggest that targeting the intracellular localization of RAS may be an alternative route to inhibit oncogenic RAS signaling in MM. Citation Format: Arnold Bolomsky, Ryan M. Young. Oncogenic RAS signals from lysosomes to activate mTORC1 in multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A037.
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Rusanescu, Gabriel, Takaya Gotoh, Xuejun Tian, and Larry A. Feig. "Regulation of Ras Signaling Specificity by Protein Kinase C." Molecular and Cellular Biology 21, no. 8 (April 15, 2001): 2650–58. http://dx.doi.org/10.1128/mcb.21.8.2650-2658.2001.
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ABSTRACT Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.
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Chakraborty, Atanu, Emily Linnane, and Sarah Ross. "Ras proteins as therapeutic targets." Biochemical Society Transactions 46, no. 5 (August 28, 2018): 1303–11. http://dx.doi.org/10.1042/bst20170529.
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Oncogenic mutations in RAS genes underlie the pathogenesis of many human tumours, and there has been intense effort for over 30 years to develop effective and tolerated targeted therapeutics for patients with Ras-driven cancers. This review summarises the progress made in Ras drug discovery, highlighting some of the recent developments in directly targeting Ras through advances in small molecule drug design and novel therapeutic strategies.
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Simanshu, Dhirendra K., and Deborah K. Morrison. "A Structure is Worth a Thousand Words: New Insights for RAS and RAF Regulation." Cancer Discovery 12, no. 4 (January 19, 2022): 899–912. http://dx.doi.org/10.1158/2159-8290.cd-21-1494.
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Abstract The RAS GTPases are frequently mutated in human cancer, with KRAS being the predominant tumor driver. For many years, it has been known that the structure and function of RAS are integrally linked, as structural changes induced by GTP binding or mutational events determine the ability of RAS to interact with regulators and effectors. Recently, a wealth of information has emerged from structures of specific KRAS mutants and from structures of multiprotein complexes containing RAS and/or RAF, an essential effector of RAS. These structures provide key insights regarding RAS and RAF regulation as well as promising new strategies for therapeutic intervention. Significance: The RAS GTPases are major drivers of tumorigenesis, and for RAS proteins to exert their full oncogenic potential, they must interact with the RAF kinases to initiate ERK cascade signaling. Although binding to RAS is typically a prerequisite for RAF to become an activated kinase, determining the molecular mechanisms by which this interaction results in RAF activation has been a challenging task. A major advance in understanding this process and RAF regulation has come from recent structural studies of various RAS and RAF multiprotein signaling complexes, revealing new avenues for drug discovery.
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Graham, Timothy E., Janet R. Pfeiffer, Rebecca J. Lee, Donna F. Kusewitt, A. Marina Martinez, Terry Foutz, Bridget S. Wilson, and Janet M. Oliver. "MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in FcεRI-Mediated Signaling." Journal of Immunology 161, no. 12 (December 15, 1998): 6733–44. http://dx.doi.org/10.4049/jimmunol.161.12.6733.
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Abstract Cross-linking the high affinity IgE receptor FcεRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the FcεRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates FcεRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to FcεRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cγ-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in FcεRI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in FcεRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.
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Therachiyil, Lubna, Anjana Anand, Abdullah Azmi, Ajaz Bhat, Hesham Korashy, and Shahab Uddin. "Role of RAS signaling in ovarian cancer." F1000Research 11 (November 4, 2022): 1253. http://dx.doi.org/10.12688/f1000research.126337.1.
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The RAS family of proteins is among the most frequently mutated genes in human malignancies. In ovarian cancer (OC), the most lethal gynecological malignancy, RAS, especially KRAS mutational status at codons 12, 13, and 61, ranges from 6–65% spanning different histo-types. Normally RAS regulates several signaling pathways involved in a myriad of cellular signaling cascades mediating numerous cellular processes like cell proliferation, differentiation, invasion, and death. Aberrant activation of RAS leads to uncontrolled induction of several downstream signaling pathways such as RAF-1/MAPK (mitogen-activated protein kinase), PI3K phosphoinositide-3 kinase (PI3K)/AKT, RalGEFs, Rac/Rho, BRAF (v-Raf murine sarcoma viral oncogene homolog B), MEK1 (mitogen-activated protein kinase kinase 1), ERK (extracellular signal-regulated kinase), PKB (protein kinase B) and PKC (protein kinase C) involved in cell proliferation as well as maintenance pathways thereby driving tumorigenesis and cancer cell propagation. KRAS mutation is also known to be a biomarker for poor outcome and chemoresistance in OC. As a malignancy with several histotypes showing varying histopathological characteristics, we focus on reviewing recent literature showcasing the involvement of oncogenic RAS in mediating carcinogenesis and chemoresistance in OC and its subtypes.
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Lightstone, Felice C., and Timothy S. Carpenter. "Abstract A015: Dynamics and lipid interactions of the RAS-RBD-CRD protein complex." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): A015. http://dx.doi.org/10.1158/1557-3125.ras23-a015.
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Abstract The high-resolution structure of the RAS G-domain complexed to the RBD and CRD domains of RAF provides critical insights into the assembly and conformation of the RAS-RAF signaling complex[1]. The influence and impact of the membrane on both the RAS and RAF proteins is a factor that we are just beginning to understand and appreciate. Molecular simulation is an ideal methodology to further study the complicated relationship between the membrane and associated proteins. Our recent work, using MuMMI[2] (Multiscale Machine-learned Modeling Infrastructure), investigated the different lipid compositions around KRAS4b and the interplay between the protein behavior and these membrane environments[3]. MuMMI uses machine learning to couple adjacent simulation scales and can be effectively scaled across some of the world’s largest high performance computing systems. The MuMMI multi-resolution framework has been expanded to include the RAF RBD-CRD domains. Here we present the overall simulation results from this latest MuMMI campaign. Tens of thousands of coarse-grained molecular dynamics simulations were completed, sampled from a variety of protein/lipid composition configurations. The orientations of the RAS-RBD-CRD complex on the membrane occupies distinct configurational states. Furthermore, the spatial patterns of lipid arrangements around these different protein states are also unique to each state. Interactions of the CRD with the membrane indicate the enrichment of very specific lipids in precise locations act to stabilize the complex. The extent, and size of the lipid ‘fingerprint’ imposed on the membrane by the RAS-RBD-CRD protein complex is significantly larger than observed for just the RAS protein on its own. We do not observe any statistically significant defined protein-protein orientations within the simulation ensemble. These observations indicate that spatial co-localize the RAS-RBD-CRD proteins in the same vicinity may be assisted by specific membrane environments. [1] Tran, Timothy H., et al. "KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation." Nature communications 12.1 (2021): 1-16. [2] Di Natale, Francesco, et al. "A massively parallel infrastructure for adaptive multiscale simulations: modeling RAS initiation pathway for cancer." Proceedings of the International Conference for High Performance Computing, Networking, Storage and Analysis. 2019. [3] Ingólfsson, Helgi I., et al. "Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins." Proceedings of the National Academy of Sciences 119.1 (2022): e2113297119. Citation Format: Felice C. Lightstone, Timothy S. Carpenter. Dynamics and lipid interactions of the RAS-RBD-CRD protein complex [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A015.
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Roy, Sandrine, Bruce Wyse, and John F. Hancock. "H-Ras Signaling and K-Ras Signaling Are Differentially Dependent on Endocytosis." Molecular and Cellular Biology 22, no. 14 (July 15, 2002): 5128–40. http://dx.doi.org/10.1128/mcb.22.14.5128-5140.2002.
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ABSTRACT Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.
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Holstein, Sarah A., Christine L. Wohlford-Lenane, and Raymond J. Hohl. "Isoprenoids Influence Expression of Ras and Ras-Related Proteins†." Biochemistry 41, no. 46 (November 2002): 13698–704. http://dx.doi.org/10.1021/bi026251x.
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Yuan, Wanqiong, and Chunli Song. "The Emerging Role of Rab5 in Membrane Receptor Trafficking and Signaling Pathways." Biochemistry Research International 2020 (February 11, 2020): 1–10. http://dx.doi.org/10.1155/2020/4186308.
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Ras analog in brain (Rab) proteins are small guanosine triphosphatases (GTPases) that belong to the Ras-like GTPase superfamily, and they can regulate vesicle trafficking. Rab proteins alternate between an activated (GTP-bound) state and an inactivated (GDP-bound) state. Early endosome marker Rab5 GTPase, a key member of the Rab family, plays a crucial role in endocytosis and membrane transport. The activated-state Rab5 recruits its effectors and regulates the internalization and trafficking of membrane receptors by regulating vesicle fusion and receptor sorting in the early endosomes. In this review, we summarize the role of small Rab GTPases Rab5 in membrane receptor trafficking and the activation of signaling pathways, such as Ras/MAPK and PI3K/Akt, which ultimately affect cell growth, apoptosis, tumorigenesis, and tumor development. This review may provide some insights for our future research and novel therapeutic targets for diseases.
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Sealover, Nancy E., and Robert L. Kortum. "Abstract 413: WT RAS signaling is an essential therapeutic target in RAS mutated cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 413. http://dx.doi.org/10.1158/1538-7445.am2022-413.
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Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS potently activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. Upstream of RAS, the RASGEFs SOS1 and SOS2 showed unique and overlapping functions to promote mutant RAS-driven transformation. SOS1 was critical for mutant RAS (G12V) activation and SOS1 inhibition augmented the efficacy of mutant RAS inhibitors. RTK−SOS2−PI3K signaling protected cells from anoikis and mediated mutant KRAS-driven transformation. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy E. Sealover, Robert L. Kortum. WT RAS signaling is an essential therapeutic target in RAS mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 413.
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Chuang, E., D. Barnard, L. Hettich, X. F. Zhang, J. Avruch, and M. S. Marshall. "Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues." Molecular and Cellular Biology 14, no. 8 (August 1994): 5318–25. http://dx.doi.org/10.1128/mcb.14.8.5318-5325.1994.
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Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.
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Chuang, E., D. Barnard, L. Hettich, X. F. Zhang, J. Avruch, and M. S. Marshall. "Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues." Molecular and Cellular Biology 14, no. 8 (August 1994): 5318–25. http://dx.doi.org/10.1128/mcb.14.8.5318.
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Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.
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Weber, Shannon, Nicole Brossier, Amanda Prechtl, Stephanie Brosius, Stephen Barnes, Landon Wilson, and Steven Carroll. "CSIG-02. R-RAS SUBFAMILY PROTEINS ELICIT DISTINCT PHYSIOLOGIC EFFECTS AND PHOSPHOPROTEOME ALTERATIONS IN NEUROFIBROMIN-NULL MPNST CELLS." Neuro-Oncology 22, Supplement_2 (November 2020): ii27—ii28. http://dx.doi.org/10.1093/neuonc/noaa215.114.
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Abstract Loss of the Ras GTPase-activating protein neurofibromin promotes the development of aggressive spindle cell neoplasms known as Malignant Peripheral Nerve Sheath Tumors (MPNSTs) in patients with the genetic disorder neurofibromatosis type 1 (NF1). Currently, the available chemotherapeutic regimens and radiotherapy are ineffective against MPNSTs, so the prognosis for patients with these neoplasms is poor. Neurofibromin loss dysregulates multiple Ras proteins in the classic (H-Ras, N-Ras, K-Ras) and R-Ras (R-Ras, R-Ras2/TC21, R-Ras3/M-Ras) subfamilies. Consequently, it is unclear which Ras proteins or pathways regulated by these Ras proteins should be therapeutically targeted in MPNSTs. We have previously shown that classic Ras proteins drive MPNST cell proliferation and survival. However, the role(s) of the R-Ras subfamily of proteins in MPNSTs have not been elucidated. To determine how R-Ras proteins contribute to the pathogenesis of neurofibromin-null MPNSTs, we introduced dominant negative (DN) R-Ras mutants, which are pan-inhibitors of the R-Ras subfamily, into MPNST cells and assessed the impact of R-Ras subfamily inhibition on mitogenesis, migration and the phosphoproteome. A panel of MPNST cell lines (STS-26T, YST-1, ST88-14, 90–8, NMS2, NMS-PC, S462, T265-2c) was used. Methodologies utilized include immunoblotting, PCR, Transwell migration, 3H-thymidine incorporation, and mass spectrometric analysis of phosphoprotein-enriched specimens. We found that R-Ras and R-Ras2 are widely expressed and can be activated in neurofibromin-null MPNST cells. In contrast to classic Ras proteins, we found that R-Ras proteins drive MPNST both mitogenesis and migration. Using mass spectrometry-based phosphoproteomics, we identified thirteen protein networks that were regulated by DN R-Ras, including networks affecting cellular movement via effects on microtubules. We chose to further study changes in ROCK1 phosphorylation and found that R-Ras subfamily proteins function, at least in some part, through the same pathways as ROCK1. We conclude that R-Ras proteins promote tumorigenesis by regulating distinct signaling pathways that regulate MPNST mitogenesis and migration.