Dafna Bar-Sagi, Ph.D.

Regulating the regulator: post-translational modification of RAS
Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.
2012 JAN ;13(1):39-51, Nature reviews. Molecular cell biology
RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation
— id: 150788, year: 2012, vol: 13, page: 39, stat: Journal Article,

Regulating the regulator: post-translational modification of RAS
Ahearn, Ian M; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R
2011 ;13(1):39-51, Nature reviews. Molecular cell biology
RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation
— id: 147707, year: 2011, vol: 13, page: 39, stat: Journal Article,

Fibronectin growth factor-binding domains are required for fibroblast survival
Lin, Fubao; Ren, Xiang-Dong; Pan, Zhi; Macri, Lauren; Zong, Wei-Xing; Tonnesen, Marcia G; Rafailovich, Miriam; Bar-Sagi, Dafna; Clark, Richard A F
2011 Jan;131(1):84-98, Journal of investigative dermatology
Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg-Gly-Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10-100 nM. FN-null cells cultured on recombinant CCBD (FNIII(8-11)) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII(8-11) contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII(8-11) and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration
— id: 138146, year: 2011, vol: 131, page: 84, stat: Journal Article,

An orthosteric inhibitor of the Ras-Sos interaction
Patgiri, Anupam; Yadav, Kamlesh K; Arora, Paramjit S; Bar-Sagi, Dafna
2011 ;7(9):585-587, Nature Chemical Biology
Mimics of alpha-helices on protein surfaces have emerged as powerful reagents for antagonizing protein-protein interactions, which are difficult to target with small molecules. Here we describe the design of a cell-permeable synthetic alpha-helix, based on the guanine nucleotide exchange factor Sos, that interferes with Ras-Sos interaction and downregulates Ras signaling in response to receptor tyrosine kinase activation
— id: 137095, year: 2011, vol: 7, page: 585, stat: Journal Article,

RAS oncogenes: weaving a tumorigenic web
Pylayeva-Gupta, Yuliya; Grabocka, Elda; Bar-Sagi, Dafna
2011 ;11(11):761-774, Nature reviews. Cancer
RAS proteins are essential components of signalling pathways that emanate from cell surface receptors. Oncogenic activation of these proteins owing to missense mutations is frequently detected in several types of cancer. A wealth of biochemical and genetic studies indicates that RAS proteins control a complex molecular circuitry that consists of a wide array of interconnecting pathways. In this Review, we describe how RAS oncogenes exploit their extensive signalling reach to affect multiple cellular processes that drive tumorigenesis
— id: 139741, year: 2011, vol: 11, page: 761, stat: Journal Article,

Distinct populations of metastases-enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra-abdominal tumor
Connolly, Michael K; Mallen-St Clair, Jon; Bedrosian, Andrea S; Malhotra, Ashim; Vera, Valery; Ibrahim, Junaid; Henning, Justin; Pachter, H Leon; Bar-Sagi, Dafna; Frey, Alan B; Miller, George
2010 Apr;87(4):713-725, Journal of leukocyte biology
The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b(+)Gr1(+) populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b(+)Gr1(+) populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b(+)Gr1(+) cells are highly suppressive of T cell activation, proliferation, and cytotoxicity and induce the development of Tregs. Moreover, liver myeloid-derived suppressor cells accelerate the development of hepatic metastases by inactivation of cytotoxic T cells. These findings may explain the propensity of patients with intra-abdominal cancers to develop liver metastases and suggest a promising target for experimental therapeutics
— id: 108918, year: 2010, vol: 87, page: 713, stat: Journal Article,

Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless
Gureasko, Jodi; Kuchment, Olga; Makino, Debora Lika; Sondermann, Holger; Bar-Sagi, Dafna; Kuriyan, John
2010 Feb 23;107(8):3430-3435, Proceedings of the National Academy of Sciences of the United States of America
Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP(2) and the local concentration of Ras-GTP, which synergize to release the DH-PH unit. Here we present a new crystal structure of SOS that contains the N-terminal histone domain in addition to the DH-PH unit and the catalytic unit (SOS(HDFC), residues 1-1049). The structure reveals that the histone domain plays a dual role in occluding the allosteric site and in stabilizing the autoinhibitory conformation of the DH-PH unit. Additional insight is provided by kinetic analysis of the activation of membrane-bound Ras by mutant forms of SOS that contain mutations in the histone and the PH domains (E108K, C441Y, and E433K) that are associated with Noonan syndrome, a disease caused by hyperactive Ras signaling. Our results indicate that the histone domain and the DH-PH unit are conformationally coupled, and that the simultaneous engagement of the membrane by a PH domain PIP(2)-binding interaction and electrostatic interactions between a conserved positively charged patch on the histone domain and the negatively charged membrane coincides with a productive reorientation of SOS at the membrane and increased accessibility of both Ras binding sites on SOS
— id: 111558, year: 2010, vol: 107, page: 3430, stat: Journal Article,

Oncogenic KRas Suppresses Inflammation-Associated Senescence of Pancreatic Ductal Cells
Lee, Kyoung Eun; Bar-Sagi, Dafna
2010 Nov 16;18(5):448-458, Cancer cell
Mutational activation of KRas is the first and most frequently detected genetic lesion in pancreatic ductal adenocarcinoma (PDAC). However, the precise role of oncogenic KRas in the pathogenesis of PDAC is not fully understood. Here, we report that the endogenous expression of oncogenic KRas suppresses premature senescence in primary pancreatic duct epithelial cells (PDEC). Oncogenic KRas-mediated senescence bypass is conferred by the upregulation of the basic helix-loop-helix transcription factor Twist that in turn abrogates p16(INK4A) induction. Moreover, the KRas-Twist-p16(INK4A) senescence bypass pathway is employed in vivo to prevent inflammation-associated senescence of pancreatic ductal epithelium. Our findings indicate that oncogenic KRas could contribute to PDAC initiation by protecting cells from entering a state of permanent growth arrest
— id: 114515, year: 2010, vol: 18, page: 448, stat: Journal Article,

Signaling endosomes: seeing is believing
Miaczynska, Marta; Bar-Sagi, Dafna
2010 Aug;22(4):535-540, Current opinion in cell biology
Signaling compartmentalization provides a highly refined mechanism to specify context-dependent cellular responses. Endosomes are an intracellular membrane-bound compartment that mediates the transport of receptor-bound signaling complexes. Owing to the development of high-resolution microscopy-based imaging techniques it has been possible to demonstrate that endosomes are actively engaged in signal reception and emission. Such observations paved the way to functional studies ascribing indispensable roles for endosomes in orchestrating signals that regulate processes such as cell migration and invasion, asymmetric cell division and differentiation, or intercellular communication
— id: 111557, year: 2010, vol: 22, page: 535, stat: Journal Article,

Perturbation of cytoskeleton dynamics by the opposing effects of Rac1 and Rac1b
Nimnual AS; Taylor LJ; Nyako M; Jeng HH; Bar-Sagi D
2010 Sep;1(2):89-97, Small GTPases
Rac1, a ubiquitously expressed member of the Rho GTPase family, plays a pivotal role in the regulation of multiple cellular processes including cytoskeleton reorganization, cell growth, differentiation and motility. Here we show that the tumor-specific splice variant of Rac1, Rac1b, negatively regulates Rac1 activity. The expression of Rac1b in HeLa cells interferes with Rac1 activation by PDGF, leads to a reduction in membrane-bound Rac1 and promotes an increase in Rho activity. The antagonistic relationship between Rac1 and Rac1b perturbs the regulatory circuitry that controls actin cytoskeleton dynamics thereby leading to tumor-linked alterations in cell morphology and motility
— id: 138351, year: 2010, vol: 1, page: 89, stat: Journal Article,

Feedback regulation of Ras signaling by Rabex-5-mediated ubiquitination
Xu, Lizhong; Lubkov, Veronica; Taylor, Laura J; Bar-Sagi, Dafna
2010 Aug 10;20(15):1372-1377, Current biology. CB
Ras proteins play a central role in transducing signals that control cell proliferation, differentiation, motility, and survival. The location-specific signaling activity of Ras has been previously shown to be regulated by ubiquitination [1]. However, the molecular machinery that controls Ras ubiquitination has not been defined. Here we demonstrate through biochemical and functional analyses that Rabex-5 (also known as RabGEF1) [2, 3] functions as an E3 ligase for Ras. Rabex-5-mediated Ras ubiquitination promotes Ras endosomal localization and leads to the suppression of ERK activation. Moreover, the Ras effector RIN1 [4, 5] is required for Rabex-5-dependent Ras ubiquitination, suggesting a feedback mechanism by which Ras activation can be coupled to ubiquitination. These findings define new elements in the regulatory circuitry that link Ras compartmentalization to signaling output
— id: 138182, year: 2010, vol: 20, page: 1372, stat: Journal Article,

Spry2 expression correlates with BRAF mutation in thyroid cancer
Xu, Lizhong; Zhou, Jun Liang; Cohen, Michael; Bar-Sagi, Dafna; Patel, Kepal N
2010 Dec;148(6):1282-1287, Surgery
BACKGROUND: BRAF mutations activate the mitogen-activated protein kinase pathway and often confer an aggressive thyroid cancer (TC) phenotype. Spry2 is an inducible negative feedback regulator of the mitogen-activated protein kinase (MAPK) pathway. The aim of this study was to investigate the role of Spry2 in TC. METHODS: TC cell lines were analyzed for Spry2 expression and MAPK pathway activation. Cells were treated with MEK inhibitor and Spry2 small hairpin RNA. Cells were analyzed for Spry2 expression and MEK/ERK phosphorylation (pMEK, pERK). Thirty human papillary TCs were analyzed for mitogen-activated protein kinase pathway activating mutations and Spry2 expression. RESULTS: Increased baseline pMEK levels and Spry2 expression was found in BRAF V600E mutant (BRAF+) cells. MEK inhibition in BRAF+ cells showed decreased Spry2 expression and decreased pMEK/pERK levels. From our tissue samples, 10 papillary TCs had BRAF mutation, and increased Spry2 expression was found only in BRAF+ tumors. CONCLUSION: Spry2 expression correlates with BRAF status in vitro and in human tissue. Spry2 may serve as a negative feedback regulator of the mitogen-activated protein kinase pathway in BRAF+ TC. Increased Spry2 expression may serve as a surrogate marker of mitogen-activated protein kinase pathway activation with prognostic and therapeutic implications
— id: 115281, year: 2010, vol: 148, page: 1282, stat: Journal Article,

Allosteric gating of Son of sevenless activity by the histone domain
Yadav, Kamlesh K; Bar-Sagi, Dafna
2010 Feb 23;107(8):3436-3440, Proceedings of the National Academy of Sciences of the United States of America
Regulated activation of Ras by receptor tyrosine kinases (RTK) constitutes a key transduction step in signaling processes that control an array of fundamental cellular functions including proliferation, differentiation, and survival. The principle mechanism by which Ras is activated down stream of RTKs involves the stimulation of guanine nucleotide exchange by the ubiquitous guanine nucleotide exchange factor Son of sevenless (Sos). In resting conditions, Sos activity is constrained by intramolecular interactions that maintain the protein in an autoinhibited conformation. Structural, biochemical, and genetic studies have implicated the histone domain (Sos-H), which comprises the most N-terminal region of Sos, in the regulation of Sos autoinhibition. However, the molecular underpinnings of this regulatory function are not well understood. In the present study we demonstrate that Sos-H possesses in vitro and in vivo membrane binding activity that is mediated, in part, by the interactions between a cluster of basic residues and phosphatidic acid. This interaction is required for Sos-dependent activation of Ras following EGF stimulation. The inducible association of Sos-H with membranes contributes to the catalytic activity of Sos by forcing the domain to adopt a conformation that destabilizes the autoinhibitory state. Thus, Sos-H plays a critical role in governing the catalytic output of Sos through the coupling of membrane recruitment to the release of autoinhibition
— id: 107379, year: 2010, vol: 107, page: 3436, stat: Journal Article,

In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-alpha
Connolly, Michael K; Bedrosian, Andrea S; Mallen-St Clair, Jon; Mitchell, Aaron P; Ibrahim, Junaid; Stroud, Andrea; Pachter, H Leon; Bar-Sagi, Dafna; Frey, Alan B; Miller, George
2009 Nov;119(11):3213-25, Journal of clinical investigation
Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-alpha. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease
— id: 105172, year: 2009, vol: 119, page: 3213, stat: Journal Article,

Ras/MAPK signaling from endomembranes
Fehrenbacher, Nicole; Bar-Sagi, Dafna; Philips, Mark
2009 Aug;3(4):297-307, Molecular oncology
Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output
— id: 101955, year: 2009, vol: 3, page: 297, stat: Journal Article,

Characterization of a Ras mutant with identical GDP- and GTP-bound structures
Ford, Bradley; Boykevisch, Sean; Zhao, Chen; Kunzelmann, Simone; Bar-Sagi, Dafna; Herrmann, Christian; Nassar, Nicolas
2009 Dec 8;48(48):11449-11457, Biochemistry
We previously characterized the G60A mutant of Ras and showed that the switch regions of the GTP-bound but not the GDP-bound form of this mutant adopt an 'open conformation' similar to that seen in nucleotide-free Ras. Here, we mutate Lys147 of the conserved (145)SAK(147) motif in the G60A background and characterize the resulting double mutant (DM). We show that RasDM is the first structure of a Ras protein with identical GDP- and GTP-bound structures. Both structures adopt the open conformation of the active form of RasG60A. The increase in the accessible surface area of the nucleotide is consistent with a 4-fold increase in its dissociation rate. Stopped-flow experiments show no major difference in the two-step kinetics of association of GDP or GTP with the wild type, G60A, or RasDM. Addition of Sos fails to accelerate nucleotide exchange. Overexpression of the G60A or double mutant of Ras in COS-1 cells fails to activate Erk and shows a strong dominant negative effect. Our data suggest that flexibility at position 60 is required for proper Sos-catalyzed nucleotide exchange and that structural information is somehow shared among the switch regions and the different nucleotide binding motifs
— id: 105530, year: 2009, vol: 48, page: 11449, stat: Journal Article,

Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas
Frank, Matthew J; Dawson, David W; Bensinger, Steven J; Hong, Jason S; Knosp, Wendy M; Xu, Lizhong; Balatoni, Cynthia E; Allen, Eric L; Shen, Rhine R; Bar-Sagi, Dafna; Martin, Gail R; Teitell, Michael A
2009 Mar 12;113(11):2478-2487, Blood
B-cell lymphoma is the most common immune system malignancy. TCL1 transgenic mice (TCL1-tg), in which TCL1 is ectopically expressed in mature lymphocytes, develop multiple B- and T-cell leukemia and lymphoma subtypes, supporting an oncogenic role for TCL1 that probably involves AKT and MAPK-ERK signaling pathway augmentation. Additional, largely unknown genetic and epigenetic alterations cooperate with TCL1 during lymphoma progression. We examined DNA methylation patterns in TCL1-tg B-cell tumors to discover tumor-associated epigenetic changes, and identified hypermethylation of sprouty2 (Spry2). Sprouty proteins are context-dependent negative or positive regulators of MAPK-ERK pathway signaling, but their role(s) in B-cell physiology or pathology are unknown. Here we show that repression of Spry2 expression in TCL1-tg mouse and human B-cell lymphomas and cell lines is associated with dense DNA hypermethylation and was reversed by inhibition of DNA methylation. Spry2 expression was induced in normal splenic B cells by CD40/B-cell receptor costimulation and regulated a negative feedback loop that repressed MAPK-ERK signaling and decreased B-cell viability. Conversely, loss of Spry2 function hyperactivated MAPK-ERK signaling and caused increased B-cell proliferation. Combined, these results implicate epigenetic silencing of Spry2 expression in B lymphoma progression and suggest it as a companion lesion to ectopic TCL1 expression in enhancing MAPK-ERK pathway signaling
— id: 95089, year: 2009, vol: 113, page: 2478, stat: Journal Article,

Raf-1 and squamous cell carcinoma: Rok-ing the boat
Grabocka, Elda; Bar-Sagi, Dafna
2009 Aug 4;16(2):85-86, Cancer cell
Squamous cell carcinoma (SCC) is the second most common form of nonmelanoma skin cancer. In this issue of Cancer Cell, Ehrenreiter et al. unveil a critical role for the Raf-1/Rok-alpha interaction in the pathogenesis of SCCs, thus paving the way for the development of therapeutic modalities to treat this malignancy
— id: 101331, year: 2009, vol: 16, page: 85, stat: Journal Article,

Abstracts of papers presented at the 2008 meeting on mechanisms and models of cancer
Bar-Sagi, Dafna; De Pinho, Ronald A; Lees, Jacqueline; Sherr, Charles J
Cold Spring Harbor NY : Cold Spring Harbor Laboratory, 2008,
— id: 2081, year: 2008, vol: , page: , stat: ,

PROLONGED EXPOSURE TO HIGH FAT DIET AND HYPERLIPIDEMIA IN MICE EXACERBATES ACUTE PANCREATITIS
Clair, JMS; Joaquin, VA; Fisher, EA; Bar-Sagi, D
2008 NOV ;37(4):482-483, Pancreas
— id: 91327, year: 2008, vol: 37, page: 482, stat: Journal Article,

Honokiol suppresses survival signals mediated by Ras-dependent phospholipase D activity in human cancer cells
Garcia, Avalon; Zheng, Yang; Zhao, Chen; Toschi, Alfredo; Fan, Judy; Shraibman, Natalie; Brown, H Alex; Bar-Sagi, Dafna; Foster, David A; Arbiser, Jack L
2008 Jul 1;14(13):4267-4274, Clinical cancer research
PURPOSE: Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. EXPERIMENTAL DESIGN: The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. RESULTS: We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. CONCLUSION: The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival
— id: 95091, year: 2008, vol: 14, page: 4267, stat: Journal Article,

Membrane-dependent signal integration by the Ras activator Son of sevenless
Gureasko, Jodi; Galush, William J; Boykevisch, Sean; Sondermann, Holger; Bar-Sagi, Dafna; Groves, Jay T; Kuriyan, John
2008 May;15(5):452-461, Nature structural & molecular biology
The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOS(cat)) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOS(cat) to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition
— id: 95093, year: 2008, vol: 15, page: 452, stat: Journal Article,

Design, synthesis, and biological evaluation of new-generation taxoids
Ojima, Iwao; Chen, Jin; Sun, Liang; Borella, Christopher P; Wang, Tao; Miller, Michael L; Lin, Songnian; Geng, Xudong; Kuznetsova, Larisa; Qu, Chuanxing; Gallager, David; Zhao, Xianrui; Zanardi, Ilaria; Xia, Shujun; Horwitz, Susan B; Mallen-St Clair, Jon; Guerriero, Jennifer L; Bar-Sagi, Dafna; Veith, Jean M; Pera, Paula; Bernacki, Ralph J
2008 Jun 12;51(11):3203-3221, Journal of medicinal chemistry
Novel second-generation taxoids with systematic modifications at the C2, C10, and C3'N positions were synthesized and their structure-activity relationships studied. A number of these taxoids exhibited exceptionally high potency against multidrug-resistant cell lines, and several taxoids exhibited virtually no difference in potency against the drug-sensitive and drug-resistant cell lines. These exceptionally potent taxoids were termed 'third-generation taxoids'. 19 (SB-T-1214), 14g (SB-T-121303), and 14i (SB-T-1213031) exhibited excellent activity against paclitaxel-resistant ovarian cancer cell lines with mutations in beta-tubulin as well, wherein the drug resistance is mediated by the beta-tubulin mutation. These taxoids were found to possess exceptional activity in promoting tubulin assembly, forming numerous very short microtubules similar to those formed by discodermolide. Taxoids 19 and 14g also showed excellent cytotoxicity against four pancreatic cancer cell lines, expressing three to four multidrug-resistant genes. Moreover, taxoid 19 exhibited excellent in vivo efficacy against highly drug-resistant CFPAC-1 pancreatic as well as DLD-1 human colon tumor xenografts in mice
— id: 95092, year: 2008, vol: 51, page: 3203, stat: Journal Article,

The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways
Qi, Jianfei; Nakayama, Koh; Gaitonde, Supriya; Goydos, James S; Krajewski, Stan; Eroshkin, Alexey; Bar-Sagi, Dafna; Bowtell, David; Ronai, Ze'ev
2008 Oct 28;105(43):16713-16718, Proceedings of the National Academy of Sciences of the United States of America
The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, our data establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms
— id: 95090, year: 2008, vol: 105, page: 16713, stat: Journal Article,

Compartmentalization-dependent ubiquitination of oncogenic Ras regulates transforming activity
Terrillon, S; Bar-Sagi, D
2008 JUN ;275(1):320-320, FEBS journal
— id: 86954, year: 2008, vol: 275, page: 320, stat: Journal Article,

Biochemical and biophysical analyses of Ras modification by ubiquitin
Terrillon, Sonia; Bar-Sagi, Dafna
2008 ;438:259-276, Methods in enzymology
Ras proteins are small GTPases that play key roles in the regulation of several cellular processes such as growth, differentiation, and transformation. Although Ras signaling was thought to occur uniformly on the inner leaflet of the plasma membrane, a growing body of evidence indicates that Ras activation happens dynamically within defined plasma membrane microdomains and at other specific intracellular compartments, thus ensuring the generation of distinct signal outputs. Yet the mechanisms that control the spatiotemporal segregation of Ras proteins remain poorly characterized. We have recently shown that the differential modification of Ras proteins by ubiquitination is a crucial factor that controls Ras intracellular trafficking and signaling potential. To better understand the process of Ras ubiquitination, it is important to establish assays that not only provide information about the nature of the ubiquitin modification involved, but also enable the monitoring of the dynamics of this process. In this chapter, we will describe biochemical and biophysical methodologies, namely immunoprecipitation, nickel-chelate affinity chromatography, and bioluminescence resonance energy transfer (BRET), for monitoring the ubiquitination of Ras proteins. Although the description focuses on Ras, the assays described can in principle be applied to the study of a range of proteins of interest that may be subject to ubiquitination, and the use of the different methods in parallel should provide new insights into the nature and dynamics of protein ubiquitination
— id: 79302, year: 2008, vol: 438, page: 259, stat: Journal Article,

Hyperlipidemia causes pancreatic inflammation and ductal proliferation in Apolipoprotein E deficient mice
Clair, JMS; Joaquin, VA; Fisher, EA; Bar-Sagi, D
2007 NOV ;35(4):414-415, Pancreas
— id: 75691, year: 2007, vol: 35, page: 414, stat: Journal Article,

Spatial Regulation of EGFR Signaling by Sprouty2
Kim, Hong Joo; Taylor, Laura J; Bar-Sagi, Dafna
2007 Mar 6;17(5):455-461, Current biology. CB
Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. Here we show that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling [1], interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization
— id: 70959, year: 2007, vol: 17, page: 455, stat: Journal Article,

Corrigendum: Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome
Tartaglia, Marco; Pennacchio, Len A; Zhao, Chen; Yadav, Kamlesh K; Fodale, Valentina; Sarkozy, Anna; Pandit, Bhaswati; Oishi, Kimihiko; Martinelli, Simone; Schackwitz, Wendy; Ustaszewska, Anna; Martin, Joel; Bristow, James; Carta, Claudio; Lepri, Francesca; Neri, Cinzia; Vasta, Isabella; Gibson, Kate; Curry, Cynthia J; Siguero, Juan Pedro Lopez; Digilio, Maria Cristina; Zampino, Giuseppe; Dallapiccola, Bruno; Bar-Sagi, Dafna; Gelb, Bruce D
2007 Feb;39(2):276-276, Nature genetics
— id: 70960, year: 2007, vol: 39, page: 276, stat: Journal Article,

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome
Tartaglia, Marco; Pennacchio, Len A; Zhao, Chen; Yadav, Kamlesh K; Fodale, Valentina; Sarkozy, Anna; Pandit, Bhaswati; Oishi, Kimihiko; Martinelli, Simone; Schackwitz, Wendy; Ustaszewska, Anna; Martin, Joel; Bristow, James; Carta, Claudio; Lepri, Francesca; Neri, Cinzia; Vasta, Isabella; Gibson, Kate; Curry, Cynthia J; Siguero, Juan Pedro Lopez; Digilio, Maria Cristina; Zampino, Giuseppe; Dallapiccola, Bruno; Bar-Sagi, Dafna; Gelb, Bruce D
2007 Jan;39(1):75-79, Nature genetics
Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development
— id: 70962, year: 2007, vol: 39, page: 75, stat: Journal Article,

Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors
Yeh, Brian J; Rutigliano, Robert J; Deb, Anrica; Bar-Sagi, Dafna; Lim, Wendell A
2007 May 31;447(7144):596-600, Nature
Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases-the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology
— id: 95094, year: 2007, vol: 447, page: 596, stat: Journal Article,

Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos
Zhao, Chen; Du, Guangwei; Skowronek, Karl; Frohman, Michael A; Bar-Sagi, Dafna
2007 Jun;9(6):706-712, Nature cell biology
The activation of Ras by the guanine nucleotide-exchange factor Son of sevenless (Sos) constitutes the rate-limiting step in the transduction process that links receptor tyrosine kinases to Ras-triggered intracellular signalling pathways. A prerequisite for the function of Sos in this context is its ligand-dependent membrane recruitment, and the prevailing model implicates both the Sos carboxy-terminal proline-rich motifs and amino-terminal pleckstrin homology (PH) domain in this process. Here, we describe a previously unrecognized pathway for the PH domain-dependent membrane recruitment of Sos that is initiated by the growth factor-induced generation of phosphatidic acid via the signalling enzyme phospholipase D2 (PLD2). Phosphatidic acid interacts with a defined site in the Sos PH domain with high affinity and specificity. This interaction is essential for epidermal growth factor (EGF)-induced Sos membrane recruitment and Ras activation. Our findings establish a crucial role for PLD2 in the coupling of extracellular signals to Sos-mediated Ras activation, and provide new insights into the spatial coordination of this activation event
— id: 95095, year: 2007, vol: 9, page: 706, stat: Journal Article,

A mouse model of hereditary pancreatitis generated by transgenic expression of R122H trypsinogen
Archer, Herbert; Jura, Natalia; Keller, James; Jacobson, Matthew; Bar-Sagi, Dafna
2006 Dec;131(6):1844-1855, Gastroenterology
BACKGROUND & AIMS: Missense mutations in human cationic trypsinogen PRSS1 are frequently detected in patients with hereditary pancreatitis, a rare genetic disease of the pancreas characterized by autodigestive necrosis, chronic inflammation, and fibrosis. To examine the link between PRSS1 mutations and the initiation and progression of hereditary pancreatitis, we have sought to generate a transgenic mouse that carries a missense mutation in the PRSS1 that is most frequently observed in patients. METHODS: A transgenic mouse was generated in which the expression of the mouse PRSS1 mutant R122H (R122H_mPRSS1) is targeted to pancreatic acinar cells by fusion to the elastase promoter. The expression of the mutant trypsinogen was assessed by immunohistochemical staining and real-time reverse transcription polymerase chain reaction analysis. The relationship between transgene expression and inflammation was analyzed by morphologic assessment of H&E-stained tissue sections, responsiveness to cerulein-induced pancreatitis, and immunohistochemical identification of cellular and biochemical components of the inflammatory response. RESULTS: Pancreata from transgenic mice display early-onset acinar cell injury and inflammatory cell infiltration. With progressing age, the transgenic mice develop pancreatic fibrosis and display acinar cell dedifferentiation. Moreover, the expression of R122H_mPRSS1 transgene is associated with enhanced response to cerulein-induced pancreatitis. Finally, cell-specific activation of the inflammation-associated signaling pathways, c-jun-N-terminal kinase and extracellular signal-regulated kinase, was observed in response to expression of R122H_mPRSS1. CONCLUSIONS: These results underscore the importance of PRSS1 mutations as pathogenic mediators of hereditary pancreatitis and indicate that persistent pancreatic injury might be causally linked to chronic pancreatitis
— id: 70963, year: 2006, vol: 131, page: 1844, stat: Journal Article,

Regulation of ras signaling dynamics by Sos-mediated positive feedback
Boykevisch, Sean; Zhao, Chen; Sondermann, Holger; Philippidou, Polyxeni; Halegoua, Simon; Kuriyan, John; Bar-Sagi, Dafna
2006 Nov 7;16(21):2173-2179, Current biology. CB
The RTK-Ras-ERK cascade is a central signaling module implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. The coupling of RTK to Ras is mediated by the Ras-specific nucleotide-exchange factor Son of Sevenless (Sos), which activates Ras by inducing the exchange of GDP for GTP . Considerable evidence indicates that the duration and amplitude of Ras signals are important determinants in controlling the biological outcome . However, the mechanisms that regulate the quantitative output of Ras signaling remain poorly understood. We define a previously unrecognized regulatory component of the machinery that specifies the kinetic properties of signals propagated through the RTK-Ras-ERK cascade. We demonstrate that the establishment of a positive feedback loop involving Ras.GTP and Sos leads to an increase in the amplitude and duration of Ras activation in response to EGF stimulation. This effect is propagated to downstream elements of the pathway as reflected by sustained EGF-induced ERK phosphorylation and enhanced SRE-dependent transcription. As a consequence, the physiological endpoint of EGF action is switched from proliferation to differentiation. We propose that the engagement of Ras/Sos positive feedback loop may contribute to the mechanism by which ligand stimulation is coupled to discrete biological responses
— id: 70964, year: 2006, vol: 16, page: 2173, stat: Journal Article,

Regulation of sprouty stability by Mnk1-dependent phosphorylation
DaSilva, John; Xu, Lizhong; Kim, Hong Joo; Miller, W Todd; Bar-Sagi, Dafna
2006 Mar;26(5):1898-1907, Molecular & cellular biology
Sprouty (Spry) proteins are negative feedback modulators of receptor tyrosine kinase pathways in Drosophila melanogaster and mammals. Mammalian Spry proteins have been shown to undergo tyrosine and serine phosphorylation in response to growth factor stimulation. While several studies have addressed the function of tyrosine phosphorylation of Spry, little is known about the significance of Spry serine phosphorylation. Here we identify mitogen-activated protein kinase-interacting kinase 1 (Mnk1) as the kinase that phosphorylates human Spry2 (hSpry2) on serines 112 and 121. Mutation of these serine residues to alanine or inhibition of Mnk1 activity increases the rate of ligand-induced degradation of hSpry2. Conversely, enhancement of serine phosphorylation achieved through either the inhibition of cellular phosphatases or the expression of active Mnk1 results in the stabilization of hSpry2. Previous studies have shown that growth factor stimulation induces the proteolytic degradation of hSpry2 by stimulating tyrosine phosphorylation on hSpry2, which in turn promotes c-Cbl binding and polyubiquitination. A mutant of hSpry2 that is deficient in serine phosphorylation displays enhanced tyrosine phosphorylation and c-Cbl binding, indicating that serine phosphorylation stabilizes hSpry2 by exerting an antagonistic effect on tyrosine phosphorylation. Moreover, loss of serine phosphorylation and the resulting enhanced degradation of hSpry2 impair its capacity to antagonize fibroblast growth factor-induced extracellular signal-regulated kinase activation. Our results imply that Mnk1-mediated serine phosphorylation of hSpry2 constitutes a regulatory mechanism to extend the temporal range of Spry activity
— id: 62919, year: 2006, vol: 26, page: 1898, stat: Journal Article,

A Ras-induced conformational switch in the Ras activator Son of sevenless
Freedman, Tanya S; Sondermann, Holger; Friedland, Gregory D; Kortemme, Tanja; Bar-Sagi, Dafna; Marqusee, Susan; Kuriyan, John
2006 Nov 7;103(45):16692-16697, Proceedings of the National Academy of Sciences of the United States of America
The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-A resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-A resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity
— id: 70965, year: 2006, vol: 103, page: 16692, stat: Journal Article,

Mapping cellular routes of Ras: a ubiquitin trail
Jura, Natalia; Bar-Sagi, Dafna
2006 Dec;5(23):2744-2747, Cell cycle
The three mammalian Ras isoforms: HRas, NRas and KRas have been widely implicated in the control of cell proliferation, survival, motility and transformation. Although nearly identical with respect to their catalytic and effector-binding properties, HRas, NRas and KRas lead to different biological outcomes in development, cell growth and cancer. This functional distinction is believed to result at least in part from the differential membrane compartmentalization of Ras isoforms. The different distribution of Ras proteins in cellular membranes dictates unique spatio-temporal patterns of activation of effector pathways. This perspective focuses on the factors that control membrane compartmentalization of Ras with an emphasis on a recently discovered novel posttranslational modification of Ras--ubiquitination. The properties of Ras ubiquitination, its contribution to the regulation of Ras intracellular trafficking and finally the influence of Ras ubiquitination on its signaling potential are discussed
— id: 70961, year: 2006, vol: 5, page: 2744, stat: Journal Article,

Differential modification of ras proteins by ubiquitination
Jura, Natalia; Scotto-Lavino, Elizabeth; Sobczyk, Aleksander; Bar-Sagi, Dafna
2006 Mar 3;21(5):679-687, Molecular cell
Ras proteins are essential components of signal transduction pathways that control cell proliferation, differentiation, and survival. It is well recognized that the functional versatility of Ras proteins is accomplished through their differential compartmentalization, but the mechanisms that control their spatial segregation are not fully understood. Here we show that HRas is subject to ubiquitin conjugation, whereas KRas is refractory to this modification. The membrane-anchoring domain of HRas is necessary and sufficient to direct the mono- and diubiquitination of HRas. Ubiquitin attachment to HRas stabilizes its association with endosomes and modulates its ability to activate the Raf/MAPK signaling pathway. Therefore, differential ubiquitination of Ras proteins may control their location-specific signaling activities
— id: 62917, year: 2006, vol: 21, page: 679, stat: Journal Article,

The role of boundary conditions in the experimental model of wound healing
Nikolic, Djordje L; Boettiger, Alistair N; Bar-Sagi, Dafna; Carbeck, Jeffrey D; Shvartsman, Stanislav Y
2006 Jul;291(1):C68-C75, American journal of physiology. Cell physiology
Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with MDCK epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied MDCK sheet migration under 3 different conditions: (1) the classic wound healing assay, (2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane (PDMS) and the monolayer is not injured but allowed to migrate upon removal of the slab, and (3) injury via PDMS membrane peel-off, where an injured monolayer migrates on plain tissue culture surface as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK 1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge
— id: 62918, year: 2006, vol: 291, page: C68, stat: Journal Article,

Pancreatic duct epithelial cell isolation and cultivation in two-dimensional and three-dimensional culture systems
Agbunag, Cristina; Lee, Kyoung Eun; Buontempo, Serena; Bar-Sagi, Dafna
2005 ;407:703-710, Methods in enzymology
Pancreatic ductal adenocarcinoma (PDA) is generally considered to have originated from pancreatic duct epithelial cells (PDEC). The ability to manipulate the growth properties of PDEC is, therefore, critical for understanding the molecular events involved in the initiation of PDA. Here, we describe methods that we have established for the isolation and maintenance of PDEC in two-dimensional and three-dimensional culture systems. The availability of these culture systems should be particularly useful for studying their relationships between specific genetic lesions and the morphogenic changes that accompany pancreatic ductal tumorigenesis
— id: 70966, year: 2005, vol: 407, page: 703, stat: Journal Article,

Structure of the G60A mutant of Ras: implications for the dominant negative effect
Ford, Bradley; Skowronek, Karlheinz; Boykevisch, Sean; Bar-Sagi, Dafna; Nassar, Nicolas
2005 Jul 8;280(27):25697-25705, Journal of biological chemistry
Substituting alanine for glycine at position 60 in v-H-Ras generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNp-bound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg(2+) ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A.GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex
— id: 62923, year: 2005, vol: 280, page: 25697, stat: Journal Article,

Chronic pancreatitis, pancreatic adenocarcinoma and the black box in-between
Jura, Natalia; Archer, Herbert; Bar-Sagi, Dafna
2005 Jan;15(1):72-77, Cell research
Pancreatic cancer is a challenging disease for patients, doctors and researchers who for decades have searched for a cure for this deadly malignancy. Although existing mouse models of pancreatic cancer have shed light on the mechanistic basis of the neoplastic conversion of the pancreas, their impact in terms of offering new diagnostics and therapeutic modalities remains limited. Chronic pancreatitis is an inflammatory disease of the pancreas that is associated with a gradual damage of the organ and an increased risk of developing neoplastic lesions. In this review, we propose that detailed studies of chronic inflammatory processes in the pancreas will provide insights into the evolution of pancreatic cancer. This information may prove useful in the design of effective therapeutic strategies to battle the disease
— id: 62924, year: 2005, vol: 15, page: 72, stat: Journal Article,

Computational docking and solution x-ray scattering predict a membrane-interacting role for the histone domain of the Ras activator son of sevenless
Sondermann, Holger; Nagar, Bhushan; Bar-Sagi, Dafna; Kuriyan, John
2005 Nov 15;102(46):16632-16637, Proceedings of the National Academy of Sciences of the United States of America
The Ras-specific nucleotide exchange factor son of sevenless (SOS) is a large, multidomain protein with complex regulation, including a Ras-dependent allosteric mechanism. The N-terminal segment of SOS, the histone domain, contains two histone folds, which is highly unusual for a cytoplasmic protein. Using a combination of computational docking, small-angle x-ray scattering, mutagenesis, and calorimetry, we show that the histone domain folds into the rest of SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and Dbl-homology (DH) domains of SOS to the catalytic domain. In this model, a positively charged surface region on the histone domain is positioned so as to provide a fourth potential anchorage site on the membrane for SOS in addition to the PH domain, the allosteric Ras molecule, and the C-terminal adapter-binding site. The histone domain in SOS interacts with the helical linker, using a region of the surface that in nucleosomes is involved in histone tetramerization. Adjacent surface elements on the histone domain that correspond to the DNA-binding surface of nucleosomes form the predicted interaction site with the membrane. The orientation and position of the histone domain in the SOS model implicates it as a potential mediator of membrane-dependent activation signals
— id: 62921, year: 2005, vol: 102, page: 16632, stat: Journal Article,

Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering
Sondermann, Holger; Zhao, Chen; Bar-Sagi, Dafna
2005 Oct;37(2):197-202, Methods
Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality
— id: 62920, year: 2005, vol: 37, page: 197, stat: Journal Article,

Ras oncogene and inflammation: partners in crime
Sparmann, Anke; Bar-Sagi, Dafna
2005 Jun;4(6):735-736, Cell cycle
It is well established that Ras oncogenes facilitate neoplastic conversion by stimulating tumor cell growth, survival and motility. However, current studies have indicated that the role of Ras in malignant transformation extends beyond these cell-intrinsic effects to include the establishment of a pro-tumorigenic host environment. We have recently demonstrated that Ras-induced secretion of the chemokine Interleukin-8 (CXCL-8/IL-8) elicits a local inflammatory reaction that is critical for neo-vascularization and sustained tumor growth. Our data identify a novel mechanism by which the Ras oncogene promotes tumor-host interactions that are essential for cancer progression, and suggest that CXCL-8 could serve as a surrogate marker for in-vivo Ras activity
— id: 62922, year: 2005, vol: 4, page: 735, stat: Journal Article,

Oncogenic K-ras drives cell cycle progression and phenotypic conversion of primary pancreatic duct epithelial cells
Agbunag, Cristina; Bar-Sagi, Dafna
2004 Aug 15;64(16):5659-5663, Cancer research
We have established a primary pancreatic duct epithelial cell culture (PDEC) system to investigate the relationship between oncogenic activation of K-ras and pancreatic ductal tumorigenesis. We have found that the acute introduction of physiological levels of oncogenic K-ras (K-rasV12) into quiescent PDECs stimulates S-phase entry and induces a pronounced increase in cell size. Both effects are dependent on the functional integrity of the phosphatidylinositol 3'-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, K-rasV12 promotes the loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin in PDEC. Our observations indicate that the oncogenic activation of K-ras is sufficient to elicit mitogenic and morphogenic responses in pancreatic ductal cells and hence is likely to play an instructive role in the initiation of pancreatic ductal adenocarcinoma
— id: 62927, year: 2004, vol: 64, page: 5659, stat: Journal Article,

Identification of the Role of MnSOD in EGFR-Positive Breast Cancer Development
Kim, Hong J; Bar-Sagi, Dafna
Ft. Belvoir Defense Technical Information Center MAY 2004,
HSpry2 has been identified as a modulator of RTK signaling pathways, and growth factor stimulation regulates hSpry2 expression and function establishing complex regulatory network. We are interested in identifying factors that control the biological activity of hSpry2. Previously our lab has shown that growth factor stimulation induces the proteasomal degradation of hSpry2 through poly-ubiquitination by E3 ubiquitin ligase, c-Cbl. Recently we found that hSpry2 is also mono-ubiquitinated. In order to characterize the function of hSpry2 mono-ubiquitination, we generated an hSpry2 ubiquitination-deficient mutant by site directed mutagenesis. Indirect immunofluorescent microscopy showed that hSpry2 ubiquitination-deficient mutant localizes to Golgi apparatus whereas wild- type hSpry2 localizes to both the Golgi and the cytoplasmic vesicular structures. Cytoplasmic vesicular localization of the mutant was regained by fusing a single ubiqutin molecule to it, strongly suggesting the involvement of mono-ubiquitination in intracellular trafficking. Taken together, these results indicate that hSpry2 monoubiquitination serves as a signal for its Golgi to endosome trafficking
— id: 2307, year: 2004, vol: , page: , stat: ,

Modulation of signalling by Sprouty: a developing story
Kim, Hong Joo; Bar-Sagi, Dafna
2004 Jun;5(6):441-450, Nature reviews. Molecular cell biology
— id: 62928, year: 2004, vol: 5, page: 441, stat: Journal Article,

Structural analysis of autoinhibition in the Ras activator Son of sevenless
Sondermann, Holger; Soisson, Stephen M; Boykevisch, Sean; Yang, Shao-Song; Bar-Sagi, Dafna; Kuriyan, John
2004 Oct 29;119(3):393-405, Cell
The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that Ras*GTP is an allosteric activator of SOS indicated that the regulation of SOS is more complex than originally envisaged. We now present crystallographic and biochemical analyses of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains and show that the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of Ras*GDP binding, and maximal activity, which requires Ras*GTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low to high activity forms as Ras*GDP is converted to Ras*GTP by SOS
— id: 62926, year: 2004, vol: 119, page: 393, stat: Journal Article,

Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis
Sparmann, Anke; Bar-Sagi, Dafna
2004 Nov;6(5):447-458, Cancer cell
The role of Ras oncogenes in promoting cellular transformation is well established. However, the contribution of Ras signaling to interactions between tumor cells and their host environment remains poorly characterized. Here, we demonstrate that the inflammatory mediator interleukin-8 (CXCL-8/IL-8) is a transcriptional target of Ras signaling. Using a tumor xenograft model, we show that Ras-dependent CXCL-8 secretion is required for the initiation of tumor-associated inflammation and neovascularization. Collectively, our data identify a novel mechanism by which the Ras oncogene can elicit a stromal response that fosters cancer progression
— id: 62925, year: 2004, vol: 6, page: 447, stat: Journal Article,

Cell phenotype specific kinetics of expression of intratracheally injected manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) during lung radioprotective gene therapy
Epperly, M W; Guo, H L; Jefferson, M; Nie, S; Gretton, J; Bernarding, M; Bar-Sagi, D; Archer, H; Greenberger, J S
2003 Jan;10(2):163-171, Gene therapy
Intratracheal (IT) injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes prior to whole lung irradiation of C57BL/6J mice provides significant protection from acute and chronic irradiation damage. We determined the duration of increased MnSOD biochemical activity and differential expression of a hemagglutinin (HA) epitope-tagged MnSOD transgene. HA-MnSOD-PL was IT injected at doses of 0-1000 microg, and mice were killed 1,2,3 or 4 days later. Other groups of mice were irradiated to 20 Gy to the pulmonary cavity 24 h after injection and killed at the same time points as non-irradiated mice. Both non-irradiated and irradiated groups of mice showed increased MnSOD biochemical activity with plasmid dose that plateaued at 100 microg of MnSOD plasmid DNA. In control mice, MnSOD biochemical activity decreased at 2, 3 or 4 days after injection. In irradiated mice, MnSOD biochemical activity decreased at day 2 but increased on days 3 and 4. HA-MnSOD expression decreased in broncheoalveolar macrophages and alveolar type-II cells 3 days after injection in non-irradiated and irradiated mice, but remained elevated in endothelial and epithelial cells past 4 days. The data provide a rationale for every second-day administration of intrapulmonary MnSOD-PL in clinical trials of radioprotective gene therapy. This should be sufficient to provide radioprotection during radiation treatments
— id: 62935, year: 2003, vol: 10, page: 163, stat: Journal Article,

Prevention of radiation-induced oral cavity mucositis by plasmid/liposome delivery of the human manganese superoxide dismutase (SOD2) transgene
Guo, Hongliang; Seixas-Silva, Jose A Jr; Epperly, Michael W; Gretton, Joan E; Shin, Dong M; Bar-Sagi, Dafna; Archer, Herbert; Greenberger, Joel S
2003 Mar;159(3):361-370, Radiation research
Oral cavity mucositis is a major toxicity of radiation therapy for head and neck cancer. In the present mouse model studies, we evaluated intraoral administration of SOD2-PL complexes 24 h before single-fraction 30-Gy irradiation for the prevention of oral cavity mucositis. Expression of the human SOD2 transgene in the oral cavity of C3H/HeNsd mice was demonstrated by nested reverse transcriptase polymerase chain reaction (RT-PCR). Mice treated intraorally with bacterial beta-galactosidase gene-plasmid/liposome (LacZ-PL) or hemagglutinin (HA)-manganese superoxide dismutase-plasmid/liposome (HA-SOD2-PL) demonstrated LacZ or HA-SOD2 expression, respectively, 24 h after injection. In a second strain of mouse, SOD2-PL-treated female athymic nude mice demonstrated significantly decreased ulceration at day 5 after 30 Gy, compared to LacZ-PL-injected, irradiated mice or irradiated controls. No further reduction in radiation-induced ulceration was detected in mice treated with both SOD2-PL and 10 mg/kg of amifostine (WR-2721) 30 min before 30 Gy compared to SOD2-PL alone. No significant protection of orthotopically transplanted murine squamous cell carcinoma (SCC-VII) tumors was detected in mice that received SOD2-PL treatment before 18 Gy. Thus overexpression of human SOD2 in the oral cavity mucosa can prevent radiation-induced mucositis with no detectable compromise in the therapeutic response of orthotopically transplanted tumors
— id: 62932, year: 2003, vol: 159, page: 361, stat: Journal Article,

hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl
Hall, Amy B; Jura, Natalia; DaSilva, John; Jang, Yeon Joo; Gong, Delquin; Bar-Sagi, Dafna
2003 Feb 18;13(4):308-314, Current biology. CB
Sprouty was originally identified in a genetic screen in Drosophila as an antagonist of fibroblast (FGF) and epidermal growth factor (EGF) signaling. Subsequently, four vertebrate homologs were discovered; among these, the human homolog Sprouty 2 (hSpry2) contains the highest degree of sequence homology to the Drosophila protein. It has been shown that hSpry2 interacts directly with c-Cbl, an E3-ubiquitin ligase, which promotes the downregulation of receptor tyrosine kinases (RTKs). In this study, we have investigated the functional consequences of the association between hSpry2 and c-Cbl. We have found that hSpry2 is ubiquitinated by c-Cbl in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of hSpry2, which in turn enhances the interaction of hSpry2 with c-Cbl. The c-Cbl-mediated ubiquitination of hSpry2 targets the protein for degradation by the 26S proteasome. An enhanced proteolytic degradation of hSpry2 is also observed in response to FGF stimulation. The FGF-induced degradation of hSpry2 limits the duration of the inhibitory effect of hSpry2 on extracellular signal-regulated kinase (ERK) activation and enables the cells to recover their sensitivity to FGF stimulation. Our results indicate that the interaction of hSpry2 with c-Cbl might serve as a mechanism for the downregulation of hSpry2 during receptor tyrosine kinase signaling
— id: 62934, year: 2003, vol: 13, page: 308, stat: Journal Article,

Suppression of Ras-stimulated transformation by the JNK signal transduction pathway
Kennedy, Norman J; Sluss, Hayla K; Jones, Stephen N; Bar-Sagi, Dafna; Flavell, Richard A; Davis, Roger J
2003 Mar 1;17(5):629-637, Genes & development
The c-Jun NH(2)-terminal kinase (JNK) phosphorylates and activates members of the activator protein-1 (AP-1) group of transcription factors and is implicated in oncogenic transformation. To examine the role of JNK, we investigated the effect of JNK deficiency on Ras-stimulated transformation. We demonstrate that although JNK does play a role in transformation in vitro, JNK is not required for tumor development in vivo. Importantly, the loss of JNK expression resulted in substantial increases in the number and growth of tumor nodules in vivo. Complementation assays demonstrated that this phenotype was caused by JNK deficiency. These data demonstrate that, in contrast to expectations, the normal function of JNK may be to suppress tumor development in vivo. This conclusion is consistent with the presence in human tumors of loss-of-function mutations in the JNK pathway
— id: 62930, year: 2003, vol: 17, page: 629, stat: Journal Article,

Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS
Margarit, S Mariana; Sondermann, Holger; Hall, Brian E; Nagar, Bhushan; Hoelz, Andre; Pirruccello, Michelle; Bar-Sagi, Dafna; Kuriyan, John
2003 Mar 7;112(5):685-695, Cell
Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras
— id: 62931, year: 2003, vol: 112, page: 685, stat: Journal Article,

Redox-dependent downregulation of Rho by Rac
Nimnual, Anjaruwee S; Taylor, Laura J; Bar-Sagi, Dafna
2003 Mar;5(3):236-241, Nature cell biology
Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases
— id: 62933, year: 2003, vol: 5, page: 236, stat: Journal Article,

Tandem histone folds in the structure of the N-terminal segment of the ras activator Son of Sevenless
Sondermann, Holger; Soisson, Stephen M; Bar-Sagi, Dafna; Kuriyan, John
2003 Dec;11(12):1583-1593, Structure
The Ras activator Son of Sevenless (Sos) contains a Cdc25 homology domain, responsible for nucleotide exchange, as well as Dbl/Pleckstrin homology (DH/PH) domains. We have determined the crystal structure of the N-terminal segment of human Sos1 (residues 1-191) and show that it contains two tandem histone folds. While the N-terminal domain is monomeric in solution, its structure is surprisingly similar to that of histone dimers, with both subunits of the histone 'dimer' being part of the same peptide chain. One histone fold corresponds to the region of Sos that is clearly similar in sequence to histones (residues 91-191), whereas the other is formed by residues in Sos (1-90) that are unrelated in sequence to histones. Residues that form a contiguous patch on the surface of the histone domain of Sos are conserved from C. elegans to humans, suggesting a potential role for this domain in protein-protein interactions
— id: 62929, year: 2003, vol: 11, page: 1583, stat: Journal Article,

Ras and Rac as activators of reactive oxygen species (ROS)
Archer, Herbert; Bar-Sagi, Dafna
2002 ;189:67-73, Methods in molecular biology
— id: 62940, year: 2002, vol: 189, page: 67, stat: Journal Article,

The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins
Egan, James E; Hall, Amy B; Yatsula, Bogdan A; Bar-Sagi, Dafna
2002 Apr 30;99(9):6041-6046, Proceedings of the National Academy of Sciences of the United States of America
Signal transduction through epidermal growth factor receptors (EGFRs) is essential for the growth and development of multicellular organisms. A genetic screen for regulators of EGFR signaling has led to the identification of Sprouty, a cell autonomous inhibitor of EGF signaling that is transcriptionally induced by the pathway. However, the molecular mechanisms by which Sprouty exerts its antagonistic effect remain largely unknown. Here we have used transient expression in human cells to investigate the functional properties of human Sprouty (hSpry) proteins. Ectopically expressed full-length hSpry1 and hSpry2 induce the potentiation of EGFR-mediated mitogen-activated protein (MAP) kinase activation. In contrast, truncation mutants of hSpry1 and hSpry2 containing the highly conserved carboxyl-terminal cysteine-rich domain inhibit EGF-induced MAP kinase activation. The potentiating effect of the full-length hSpry2 proteins on EGF signaling is mediated by the amino-terminal domain and results from the sequestration of c-Cbl, which in turn leads to the inhibition of EGFR ubiquitination and degradation. These results indicate that hSpry2 can function both as a negative and positive regulator of EGFR-mediated MAP kinase signaling in a domain-dependent fashion. A dual function of this kind could provide a mechanism for achieving proper balance between the activation and repression of EGFR signaling
— id: 62943, year: 2002, vol: 99, page: 6041, stat: Journal Article,

Pulmonary irradiation-induced expression of VCAM-I and ICAM-I is decreased by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy
Epperly, Michael W; Sikora, Christine A; DeFilippi, Stacy J; Gretton, Joan E; Bar-Sagi, Dafna; Archer, Herbert; Carlos, Timothy; Guo, HongLiang; Greenberger, Joel S
2002 ;8(4):175-187, Biology of blood & marrow transplantation
Pulmonary toxicity is a major complication of total body irradiation used in preparation of patients for bone marrow transplantation. The mechanism of the late pulmonary damage manifested by fibrosis is unknown. In C57BL/6NHsd mice, manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intratracheal injection 24 hours prior to 20 Gy single-fraction irradiation to both lungs significantly reduced late irradiation damage. Single intratracheal injections of MnSOD-PL, at concentrations as low as 250 microg of plasmid DNA, in a constant volume of 78 microL of liposomes, reduced late damage. To determine whether a slowly proliferating population of cells in the lung was responsible for initiation of fibrosis and was altered by MnSOD-PL therapy, 20 Gy total lung-irradiated mice were examined at serial time points for bromodeoxyuridine (BrdU) uptake in sites of cell division. There was low-level, but nonsignificant, increased cell proliferation detected at 80 days, with a significant increase at 100 days, 120 days, and at the time of death. Immunohistochemical assay for up-regulation of adhesion molecules associated with recruitment, transendothelial migration, and proliferation of bronchoalveolar macrophages revealed significant up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) at 100 days with further increases up to the time of death. Increases were first detected in endothelin-positive endothelial cells. MnSOD-PL administration prior to irradiation decreased both BrdU incorporation and delayed expression of VCAM-1 and ICAM-1. The data indicate that the appearance of late irradiation-induced pulmonary fibrosis is associated with the up-regulation of adhesion molecules and suggest that potential targets for intervention may focus on the pulmonary vascular endothelium
— id: 62942, year: 2002, vol: 8, page: 175, stat: Journal Article,

The pleckstrin homology (PH) domain-interacting protein couples the insulin receptor substrate 1 PH domain to insulin signaling pathways leading to mitogenesis and GLUT4 translocation
Farhang-Fallah, Janet; Randhawa, Varinder K; Nimnual, Anjaruwee; Klip, Amira; Bar-Sagi, Dafna; Rozakis-Adcock, Maria
2002 Oct;22(20):7325-7336, Molecular & cellular biology
Receptor-mediated tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) is required for the propagation of many of insulin's biological effects. The amino-terminal pleckstrin homology (PH) domain of IRS-1 plays a pivotal role in promoting insulin receptor (IR)-IRS-1 protein interactions. We have recently reported the isolation of a PH domain-interacting protein, PHIP, which selectively binds to the IRS-1 PH domain and is stably associated with IRS-1 in mammalian cells. Here we demonstrate that overexpression of PHIP in fibroblasts enhances insulin-induced transcriptional responses in a mitogen-activated protein kinase-dependent manner. In contrast, a dominant-negative mutant of PHIP (DN-PHIP) was shown to specifically block transcriptional and mitogenic signals elicited by insulin and not serum. In order to examine whether PHIP/IRS-1 complexes participate in the signal transduction pathway linking the IR to GLUT4 traffic in muscle cells, L6 myoblasts stably expressing a myc-tagged GLUT4 construct (L6GLUT4myc) were transfected with either wild-type or dominant-interfering forms of PHIP. Whereas insulin-dependent GLUT4myc membrane translocation was not affected by overexpression of PHIP, DN-PHIP caused a nearly complete inhibition of GLUT4 translocation, in a manner identical to that observed with a dominant-negative mutant of the p85 subunit of phosphatidylinositol 3-kinase (Deltap85). Furthermore, DN-PHIP markedly inhibited insulin-stimulated actin cytoskeletal reorganization, a process required for the productive incorporation of GLUT4 vesicles at the cell surface in L6 cells. Our results are consistent with the hypothesis that PHIP represents a physiological protein ligand of the IRS-1 PH domain, which plays an important role in insulin receptor-mediated mitogenic and metabolic signal transduction
— id: 62937, year: 2002, vol: 22, page: 7325, stat: Journal Article,

The structural basis for the transition from Ras-GTP to Ras-GDP
Hall, Brian E; Bar-Sagi, Dafna; Nassar, Nicolas
2002 Sep 17;99(19):12138-12142, Proceedings of the National Academy of Sciences of the United States of America
The conformational changes in Ras that accompany the hydrolysis of GTP are critical to its function as a molecular switch in signaling pathways. Understanding how GTP is hydrolyzed by revealing the sequence of intermediary structures in the reaction is essential for understanding Ras signaling. Until now, no structure of an intermediate in GTP hydrolysis has been experimentally determined for Ras alone. We have solved the crystal structure of the Ala-59 to Gly mutant of Ras, (RasA59G), bound to guanosine 5'-imidotriphosphate or GDP to 1.7-A resolution. In the guanosine 5'-imidotriphosphate-bound form, this mutant adopts a conformation that is intermediate between the GTP- and GDP-bound forms of wild-type Ras and that is similar to what has been predicted by molecular dynamics simulation [Ma, J. P. & Karplus, M. (1997) Proc. Natl. Acad. Sci. USA 94, 11905-11910]. This conformation is stabilized by direct and water-mediated interactions between the switch 1 and switch 2 regions and is characterized by an increase in the binding affinity for GTP. We propose that the structural changes promoted by the Ala-59 to Gly mutation exhibit a discrete conformational state assumed by wild-type Ras during GTP hydrolysis
— id: 62938, year: 2002, vol: 99, page: 12138, stat: Journal Article,

Autoinhibition of Sos by intramolecular interactions
Hall, Brian E; Yang, Shao-Song; Bar-Sagi, Dafna
2002 Jan 1;7:d288-d294, Frontiers in biosciences
Sos proteins function as activators of Ras signaling by catalyzing guanine nucleotide exchange on Ras. Sos regulation was initially thought to be accomplished primarily through its growth factor-dependent recruitment to the plasma membrane. More recent data has indicated that while membrane association is an indispensable means of Sos regulation, additional mechanisms involving intramolecular interactions function to control Sos activity towards Ras. This review will examine the experimental evidence for Sos intramolecular interactions and their contribution to Sos regulation
— id: 62944, year: 2002, vol: 7, page: d288, stat: Journal Article,

The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase
Lei, Kui; Nimnual, Anjaruwee; Zong, Wei-Xing; Kennedy, Norman J; Flavell, Richard A; Thompson, Craig B; Bar-Sagi, Dafna; Davis, Roger J
2002 Jul;22(13):4929-4942, Molecular & cellular biology
Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis
— id: 62941, year: 2002, vol: 22, page: 4929, stat: Journal Article,

Direct coupling of the cell cycle and cell death machinery by E2F
Nahle, Zaher; Polakoff, Julia; Davuluri, Ramana V; McCurrach, Mila E; Jacobson, Matthew D; Narita, Masashi; Zhang, Michael Q; Lazebnik, Yuri; Bar-Sagi, Dafna; Lowe, Scott W
2002 Nov;4(11):859-864, Nature cell biology
Unrestrained E2F activity forces S phase entry and promotes apoptosis through p53-dependent and -independent mechanisms. Here, we show that deregulation of E2F by adenovirus E1A, loss of Rb or enforced E2F-1 expression results in the accumulation of caspase proenzymes through a direct transcriptional mechanism. Increased caspase levels seem to potentiate cell death in the presence of p53-generated signals that trigger caspase activation. Our results demonstrate that mitogenic oncogenes engage a tumour suppressor network that functions at multiple levels to efficiently induce cell death. The data also underscore how cell cycle progression can be coupled to the apoptotic machinery
— id: 62936, year: 2002, vol: 4, page: 859, stat: Journal Article,

The two hats of SOS
Nimnual, Anjaruwee; Bar-Sagi, Dafna
2002 Aug 13;2002(145):PE36-PE36, Science's STKE
Son of sevenless (SOS) is a guanine nucleotide exchange factor that activates Ras in response to growth factor stimulation. SOS also appears to serve as a guanine nucleotide exchanger for Rac and, thus, may be involved in cytoskeleton reorganization. Nimnual and Bar-Sagi discuss how these two activities of SOS can be regulated and how SOS may be recruited to different cellular locations through interactions with the adaptor proteins Grb2 and E3b1
— id: 62939, year: 2002, vol: 2002, page: PE36, stat: Journal Article,

A Ras by any other name
Bar-Sagi D
2001 Mar;21(5):1441-1443, Molecular & cellular biology
— id: 62950, year: 2001, vol: 21, page: 1441, stat: Journal Article,

Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) administration protects mice from esophagitis associated with fractionated radiation
Epperly MW; Kagan VE; Sikora CA; Gretton JE; Defilippi SJ; Bar-Sagi D; Greenberger JS
2001 Aug 20;96(4):221-231, International journal of cancer
Intraesophageal administration of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD-PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD-PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial beta-galactosidase gene)-plasmid/liposome (LacZ-PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope-tagged MnSOD (HA-MnSOD). In fractionation protocols, mice receiving MnSOD-PL, but not LacZ-PL (200 microl of plasmid/liposomes containing 200 microg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD-PL administration. The HA-MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase-plasmid/liposome (GPX-PL), or liposomes containing MnSOD protein, vitamin E, co-enzyme Q10, or 21-aminosteroid. Thus, MnSOD-PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD-PL treatment into human esophageal radiation protection
— id: 62946, year: 2001, vol: 96, page: 221, stat: Journal Article,

Rac1 mediates STAT3 activation by autocrine IL-6
Faruqi TR; Gomez D; Bustelo XR; Bar-Sagi D; Reich NC
2001 Jul 31;98(16):9014-9019, Proceedings of the National Academy of Sciences of the United States of America
The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (Delta1-187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-kappaB activation or disruption of IL-6-mediated signaling through the expression of IkappaBalpha S32AS36A and suppressor of cytokine signaling 3, respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase/STAT signaling
— id: 62947, year: 2001, vol: 98, page: 9014, stat: Journal Article,

Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange
Hall BE; Yang SS; Boriack-Sjodin PA; Kuriyan J; Bar-Sagi D
2001 Jul 20;276(29):27629-27637, Journal of biological chemistry
Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation
— id: 62948, year: 2001, vol: 276, page: 27629, stat: Journal Article,

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling
Lu W; Gong D; Bar-Sagi D; Cole PA
2001 Oct;8(4):759-769, Molecular cell
The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using 'expressed protein ligation', nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction
— id: 62945, year: 2001, vol: 8, page: 759, stat: Journal Article,

Differential activation of the Rac pathway by Ha-Ras and K-Ras
Walsh AB; Bar-Sagi D
2001 May 11;276(19):15609-15615, Journal of biological chemistry
Ras proteins are key regulators of cell growth and differentiation. Mammalian cells express three closely related Ras proteins: Ha-Ras, K-Ras, and N-Ras. We have compared the abilities of the Ha-Ras and K-Ras isoforms to activate the Rac effector pathway, using three Rac-dependent readouts: induction of membrane ruffling and pinocytosis, stimulation of cell motility, and Pak binding. The total surface area of membrane ruffles induced by K-RasV12 was 2-fold greater than that induced by Ha-RasV12. Likewise, the number of K-RasV12-induced pinocytic vesicles per cell was approximately 2-fold greater than that induced by Ha-RasV12. In a wound healing assay, K-RasV12-injected cells migrated twice as fast as Ha-RasV12-injected cells. Moreover, the Pak binding activity of Rac, which is indicative of the amount of GTP-bound Rac, was higher in K-RasV12-expressing cells than Ha-RasV12-expressing cells. These results suggest that K-Ras activates Rac more efficiently than Ha-Ras. The preferential activation of Rac by K-Ras is dependent on the mode of membrane anchoring and impacts on the ability of K-Ras to regulate cell survival
— id: 62949, year: 2001, vol: 276, page: 15609, stat: Journal Article,

Ras and Rho GTPases: a family reunion
Bar-Sagi D; Hall A
2000 Oct 13;103(2):227-238, Cell
— id: 62951, year: 2000, vol: 103, page: 227, stat: Journal Article,

Induction of the cellular E2F-1 promoter by the adenovirus E4-6/7 protein
Schaley J; O'Connor RJ; Taylor LJ; Bar-Sagi D; Hearing P
2000 Mar;74(5):2084-2093, Journal of virology
The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression
— id: 62955, year: 2000, vol: 74, page: 2084, stat: Journal Article,

Single cell assays for Rac activity
Taylor LJ; Walsh AB; Hearing P; Bar-Sagi D
2000 ;325:327-334, Methods in enzymology
— id: 62952, year: 2000, vol: 325, page: 327, stat: Journal Article,

Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway
Tournier C; Hess P; Yang DD; Xu J; Turner TK; Nimnual A; Bar-Sagi D; Jones SN; Flavell RA; Davis RJ
2000 May 5;288(5467):870-874, Science
The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnk genes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic signal transduction through the JNK pathway
— id: 62953, year: 2000, vol: 288, page: 870, stat: Journal Article,

Workshop on specificity in Ras and Rho-mediated signalling events
Bos JL; Lacal, Juan Carlos; Hall, A; Bar-Sagi, D
[Madrid] : Instituto Juan March de Estudios e Investigaciones, 1999,
— id: 2203, year: 1999, vol: , page: , stat: ,

Suppression of Ras-induced apoptosis by the Rac GTPase
Joneson T; Bar-Sagi D
1999 Sep;19(9):5892-5901, Molecular & cellular biology
Ras is an essential component of signal transduction pathways that control cell proliferation, differentiation, and survival. In this study we have examined the cellular responses to high-intensity Ras signaling. Expression of increasing amounts of the oncogenic form of human HRas, HRasV12, results in a dose-dependent induction of apoptosis in both primary and immortalized cells. The induction of apoptosis by HRasV12 is blocked by activated Rac and potentiated by dominant interfering Rac. The ability of Rac to suppress Ras-induced apoptosis is dependent on effector pathway(s) controlled by the insert region and is linked to the activation of NF-kappaB. The apoptotic effect of HRasV12 requires the activation of both the ERK and JNK mitogen-activated protein kinase cascade and is independent of p53. These results demonstrate a role for Rac in controlling signals that are necessary for cell survival, and suggest a mechanism by which Rac activity can confer growth advantage to cells transformed by the ras oncogene
— id: 62957, year: 1999, vol: 19, page: 5892, stat: Journal Article,

SCH 51344, an inhibitor of RAS/RAC-mediated cell morphology pathway
Kumar CC; Ohashi K; Nagata K; Walsh A; Bar-Sagi D; Mizuno K
1999 ;886:122-131, Annals of the New York Academy of Sciences
RAS interacts with multiple targets in the cell and controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. These two pathways appear to act synergistically to cause transformation. Human smooth muscle alpha-actin promoter is repressed in RAS-transformed cells and derepressed in revertant cell lines, suggesting that it is a sensitive marker to follow phenotypic changes in fibroblast cells. SCH 51344 is a pyrazoloquinoline derivative identified on the basis of its ability to derepress alpha-actin promoter in RAS-transformed cells. Previous studies have shown that SCH 51344 is a potent inhibitor of RAS transformation. However, SCH 51344 had very little effect on the activities of proteins in the ERK pathway, suggesting that it inhibits RAS transformation by a novel mechanism. Recently, we have demonstrated that SCH 51344 specifically blocks membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS, and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and JUN kinase activities. SCH 51344 was effective in inhibiting the anchorage-independent growth of Rat-2 fibroblast cells transformed by the three forms of oncogenic RAS and RAC V12. These results indicate that SCH 51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting this pathway may be an effective approach to inhibiting transformation by RAS and other oncogenes
— id: 62954, year: 1999, vol: 886, page: 122, stat: Journal Article,

Nucleolar Arf sequesters Mdm2 and activates p53
Weber JD; Taylor LJ; Roussel MF; Sherr CJ; Bar-Sagi D
1999 May;1(1):20-26, Nature cell biology
The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation
— id: 62956, year: 1999, vol: 1, page: 20, stat: Journal Article,

Transmembrane signaling protocols
Bar-Sagi, Dafna
Totowa NJ : Humana Press, 1998,
— id: 1087, year: 1998, vol: , page: , stat: ,

The structural basis of the activation of Ras by Sos
Boriack-Sjodin PA; Margarit SM; Bar-Sagi D; Kuriyan J
1998 Jul 23;394(6691):337-343, Nature
The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. First, the insertion into Ras of an alpha-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. Second, side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras-Sos complex adopts a structure that allows nucleotide release and rebinding
— id: 62959, year: 1998, vol: 394, page: 337, stat: Journal Article,

Regulation of Sos activity by intramolecular interactions
Corbalan-Garcia S; Margarit SM; Galron D; Yang SS; Bar-Sagi D
1998 Feb;18(2):880-886, Molecular & cellular biology
The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity
— id: 62963, year: 1998, vol: 18, page: 880, stat: Journal Article,

A Rac1 effector site controlling mitogenesis through superoxide production
Joneson T; Bar-Sagi D
1998 Jul 17;273(29):17991-17994, Journal of biological chemistry
The Rac GTP-binding protein controls signal transduction pathways that are critical for mitogenesis and oncogenesis (1,2). The biochemical nature of these signaling pathways is presently unknown. Here we report that a region in Rac1 (residues 124-135), previously defined as the insert region (3), is essential for its mitogenic activity. Deletion of this region does not interfere with the ability of Rac1 to induce cytoskeletal changes or to activate the Jun kinase mitogen-activated protein kinase cascade but abrogates Rac1-induced stimulation of DNA synthesis and Rac1-mediated superoxide production in quiescent fibroblasts. Treatment of cells with agents that abolish superoxide generation inhibits specifically the mitogenic effect of Rac1. Our results identify an effector site in Rac1 that is necessary for mitogenic signaling and implicate superoxide generation as a candidate effector pathway of Rac1-dependent cell growth
— id: 62960, year: 1998, vol: 273, page: 17991, stat: Journal Article,

Kinase suppressor of Ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated Ras, and Ras effectors
Joneson T; Fulton JA; Volle DJ; Chaika OV; Bar-Sagi D; Lewis RE
1998 Mar 27;273(13):7743-7748, Journal of biological chemistry
Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development
— id: 62961, year: 1998, vol: 273, page: 7743, stat: Journal Article,

Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos
Nimnual AS; Yatsula BA; Bar-Sagi D
1998 Jan 23;279(5350):560-563, Science
The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways
— id: 62962, year: 1998, vol: 279, page: 560, stat: Journal Article,

Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein
Soisson SM; Nimnual AS; Uy M; Bar-Sagi D; Kuriyan J
1998 Oct 16;95(2):259-268, Cell
Proteins containing Dbl homology (DH) domains activate Rho-family GTPases by functioning as specific guanine nucleotide exchange factors. All known DH domains have associated C-terminal pleckstrin homology (PH) domains that are implicated in targeting and regulatory functions. The crystal structure of a fragment of the human Son of sevenless protein containing the DH and PH domains has been determined at 2.3 A resolution. The entirely alpha-helical DH domain is unrelated in architecture to other nucleotide exchange factors. The active site of the DH domain, identified on the basis of sequence conservation and structural features, lies near the interface between the DH and PH domains. The structure suggests that ligation of the PH domain will be coupled structurally to the GTPase binding site
— id: 62958, year: 1998, vol: 95, page: 259, stat: Journal Article,

The role of the PH domain in the signal-dependent membrane targeting of Sos
Chen RH; Corbalan-Garcia S; Bar-Sagi D
1997 Mar 17;16(6):1351-1359, EMBO journal
The pleckstrin homology (PH) domain is a conserved protein module present in diverse signal transducing proteins. To investigate the function of the PH domain of the Ras exchanger Sos, we have generated a recombinant (His)6-tagged PH domain from human Sos1 (PH-Sos). Here we show that PH-Sos binds with high affinity(1.5 microM) to lipid vesicles containing the negatively charged phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). When microinjected into serum-deprived rat embryo fibroblasts or COS cells, PH-Sos displays a homogenous subcellular distribution. However, PH-Sos rapidly accumulates in the plasma membrane following serum stimulation and, under these conditions, is localized preferentially to the leading edge of motile cells. Surprisingly, the membrane localization of PH-Sos is not dependent on its ability to bind PIP2. Overexpression of the PH domain of Sos has a pronounced dominant-negative effect on serum-induced activation of the Ras signaling pathway. These results suggest that the PH domain of Sos participates in regulating the inducible association of Sos with the membrane, and indicate the presence of specific ligands that interact with this domain to bring about the activation of Ras
— id: 62967, year: 1997, vol: 16, page: 1351, stat: Journal Article,

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization
Colley WC; Sung TC; Roll R; Jenco J; Hammond SM; Altshuller Y; Bar-Sagi D; Morris AJ; Frohman MA
1997 Mar 1;7(3):191-201, Current biology. CB
BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2
— id: 62968, year: 1997, vol: 7, page: 191, stat: Journal Article,

Ras effectors and their role in mitogenesis and oncogenesis
Joneson T; Bar-Sagi D
1997 Aug;75(8):587-593, Journal of Molecular Medicine (Berlin)
Ras proteins are membrane-bound GTP-binding proteins that play a critical role in the control of cell growth. Through a large number of genetic and biochemical studies it is becoming increasingly evident that the biological activity of Ras proteins is mediated by multiple signaling pathways. This review provides an account of the target proteins that interact with Ras and the functional consequences of these interactions. The relative contribution of the different Ras effector pathways to the mitogenic and oncogenic effects of Ras are discussed
— id: 62966, year: 1997, vol: 75, page: 587, stat: Journal Article,

A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway
Kouhara H; Hadari YR; Spivak-Kroizman T; Schilling J; Bar-Sagi D; Lax I; Schlessinger J
1997 May 30;89(5):693-702, Cell
Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. In this report, we describe the purification, cloning, and characterization of a novel protein, designated FRS2, that is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. We find that FRS2 is myristylated and that this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. Finally, we demonstrate that FRS2 is closely related and probably indentical to SNT, the long-sought target of FGF and NGF receptors
— id: 56961, year: 1997, vol: 89, page: 693, stat: Journal Article,

SCH 51344-induced reversal of RAS-transformation is accompanied by the specific inhibition of the RAS and RAC-dependent cell morphology pathway
Walsh AB; Dhanasekaran M; Bar-Sagi D; Kumar CC
1997 Nov 20;15(21):2553-2560, Oncogene
RAS interacts with multiple targets in the cell and controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. These two pathways appear to act synergistically to cause transformation. SCH 51344 is a pyrazolo-quinoline derivative identified based on its ability to derepress transformation sensitive alpha-actin promoter in RAS-transformed cells. Previous studies have shown that SCH 51344 is a potent inhibitor of RAS-transformation. However, SCH 51344 had very little effect on the activities of proteins in the ERK pathway, suggesting that it inhibits RAS-transformation by a novel mechanism. In this study, we show that SCH 51344 specifically blocks membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and JUN kinase activities. SCH 51344 was effective in inhibiting the anchorage-independent growth of Rat-2 fibroblast cells transformed by the three forms of oncogenic RAS and RAC V12. These results indicate that SCH 51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting this pathway may be an effective approach to inhibit transformation by RAS and other oncogenes
— id: 62965, year: 1997, vol: 15, page: 2553, stat: Journal Article,

The solution structure of the pleckstrin homology domain of human SOS1. A possible structural role for the sequential association of diffuse B cell lymphoma and pleckstrin homology domains
Zheng J; Chen RH; Corblan-Garcia S; Cahill SM; Bar-Sagi D; Cowburn D
1997 Nov 28;272(48):30340-30344, Journal of biological chemistry
A large subset of pleckstrin homology (PH) domains are immediately to the C terminus of diffuse B cell lymphoma (Dbl) homology (DbH) domains. Dbl domains are generally considered to be GTPase-exchange factors; many are proto-oncogenes. PH domains appear to function as membrane-recruitment factors, or have specific protein-protein interactions. Since dual domain (DbH/PH) constructs are known to have significant properties in other pathways, it is possible that a defined interdomain relationship is required for DbH/PH function. We determined the solution structure of the human SOS1 PH domain for a construct partially extended into the preceding DbH domain. There are specific structural contacts between the PH and the vestigial DbH domain. This appears to involve structural elements common to this subfamily of PH domains, and to DbH domains. The human SOS1 PH domain binds to inositol 1,4,5-triphosphate with a approximately 60 mu M affinity. Using chemical shift titration, the binding site is identified to be essentially identical to that observed crystallographically for the inositol 1,4,5-triphosphate complex with the PH domain of phospholipase Cdelta. This site may serve as an interdomain regulator of DbH or other domains' functions. While the overall fold of the human SOS1 PH domain is similar to other PH domains, the size and position of the intrastrand loops and the presence of an N-terminal alpha-helix of the vestigial DbH domain suggest that the subfamily of PH domains associated with DbH domains may be a well defined structural group in which the PH domain is a membrane recruiter and modulator
— id: 62964, year: 1997, vol: 272, page: 30340, stat: Journal Article,

Insulin-induced dissociation of Sos from Grb2 does not contribute to the down regulation of Ras activation
Corbalan-Garcia S; Degenhardt KR; Bar-Sagi D
1996 Mar 7;12(5):1063-1068, Oncogene
Activation of Ras by a number of receptor tyrosine kinases is mediated by the guanine nucleotide exchange factor Sos. This activation is thought to occur as a result of the recruitment to the plasma membrane of a complex consisting of Sos and the adaptor molecule Grb2. Growth factor stimulation has been shown to induce the rapid phosphorylation of Sos on serine and threonine residues. In rat L6 cells, insulin-induced Sos phosphorylation is accompanied by a partial dissociation of the Grb2-Sos complex. In this study we have investigated the relationship between Sos phosphorylation and Grb2 association. To this end, we have utilized cAMP because it has been demonstrated that elevation of cytoplasmic levels of cAMP inhibits growth factor-induced Sos phosphorylation. We show that in rat L6 cells, cAMP treatment prevents both the insulin-stimulated Sos phosphorylation and Grb2 dissociation. However, cAMP treatment has no effect on the duration of insulin-induced Ras activation. These results suggest that the kinetics of Ras activation are independent of the phosphorylation-induced dissociation of Sos from Grb2
— id: 62973, year: 1996, vol: 12, page: 1063, stat: Journal Article,

Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2
Corbalan-Garcia S; Yang SS; Degenhardt KR; Bar-Sagi D
1996 Oct;16(10):5674-5682, Molecular & cellular biology
The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation
— id: 62970, year: 1996, vol: 16, page: 5674, stat: Journal Article,

RAC regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase
Joneson T; McDonough M; Bar-Sagi D; Van Aelst L
1996 Nov 22;274(5291):1374-1376, Science
The RAC guanine nucleotide binding proteins regulate multiple biological activities, including actin polymerization, activation of the Jun kinase (JNK) cascade, and cell proliferation. RAC effector loop mutants were identified that separate the ability of RAC to interact with different downstream effectors. One mutant of activated human RAC protein, RACV12H40 (with valine and histidine substituted at position 12 and 40, respectively), was defective in binding to PAK3, a Ste20-related p21-activated kinase (PAK), but bound to POR1, a RAC-binding protein. This mutant failed to stimulate PAK and JNK activity but still induced membrane ruffling and mediated transformation. A second mutant, RACV12L37 (with leucine substituted at position 37), which bound PAK but not POR1, induced JNK activation but was defective in inducing membrane ruffling and transformation. These results indicate that the effects of RAC on the JNK cascade and on actin polymerization and cell proliferation are mediated by distinct effector pathways that diverge at the level of RAC itself
— id: 62969, year: 1996, vol: 274, page: 1374, stat: Journal Article,

Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS
Joneson T; White MA; Wigler MH; Bar-Sagi D
1996 Feb 9;271(5250):810-812, Science
The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS
— id: 62974, year: 1996, vol: 271, page: 810, stat: Journal Article,

The Ras superfamily of GTPases
Macara IG; Lounsbury KM; Richards SA; McKiernan C; Bar-Sagi D
1996 Apr;10(5):625-630, FASEB journal
The Ras superfamily of small GTPases comprises a group of molecular switches that regulate an astonishing diversity of cellular functions. A deep understanding of mitogenesis, cytoskeletal organization, vesicle traffic, and nuclear transport now requires the inclusion of the small GTPases as essential components of the molecular machines that drive these processes. The rich complexity of the control mechanisms involved is evidenced by the recent discoveries of GTPase cascades, multiple downstream effectors, and interconnected networks of GTPase-regulated protein kinase cascades. The 1995 FASEB Summer Conference at Snowmass Village, Colorado, on the Ras GTPase superfamily provided testimony to the broad impact that the study of these proteins continues to exert on cell biology
— id: 62972, year: 1996, vol: 10, page: 625, stat: Journal Article,

Identification of a novel Rac1-interacting protein involved in membrane ruffling
Van Aelst L; Joneson T; Bar-Sagi D
1996 Aug 1;15(15):3778-3786, EMBO journal
The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways
— id: 62971, year: 1996, vol: 15, page: 3778, stat: Journal Article,

Mammalian cell microinjection assay
Bar-Sagi D
1995 ;255:436-442, Methods in enzymology
— id: 62977, year: 1995, vol: 255, page: 436, stat: Journal Article,

Purification of baculovirus-expressed human Sos1 protein
Frech M; Cussac D; Chardin P; Bar-Sagi D
1995 ;255:125-129, Methods in enzymology
— id: 62978, year: 1995, vol: 255, page: 125, stat: Journal Article,

Inhibition of ras-induced proliferation and cellular transformation by p16INK4
Serrano M; Gomez-Lahoz E; DePinho RA; Beach D; Bar-Sagi D
1995 Jan 13;267(5195):249-252, Science
The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocked entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block was relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppressed cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth
— id: 62976, year: 1995, vol: 267, page: 249, stat: Journal Article,

Differential interactions of human Sos1 and Sos2 with Grb2
Yang SS; Van Aelst L; Bar-Sagi D
1995 Aug 4;270(31):18212-18215, Journal of biological chemistry
The guanine nucleotide exchange factor Son of sevenless (Sos) performs a crucial step in the coupling of receptor tyrosine kinases to Ras activation. Mammalian cells contain two related but distinct Sos proteins, Sos1 and Sos2. Although they share a high degree of overall similarity, it is not known to what extent their biological and biochemical properties overlap. In the present study, we have compared the interactions of the two human homologues of Sos, hSos1 and hSos2, with the adaptor protein Grb2. We show that hSos2 interacts with Grb2 via its proline-rich COOH-terminal domain and that this interaction is dependent on the SH3 domains of Grb2. In general, these characteristics are similar to the ones reported previously for the interaction of hSos1 with Grb2. However, the apparent binding affinity of hSos2 for Grb2 is significantly higher relative to that of hSos1 both in vitro and in vivo. The region conferring this higher binding affinity has been mapped to residues 1126-1242 of the hSos2 COOH-terminal domain. These results suggest that Sos1 and Sos2 may differentially contribute to receptor-mediated Ras activation
— id: 62975, year: 1995, vol: 270, page: 18212, stat: Journal Article,

The Sos (Son of sevenless) protein
Bar-Sagi, D
1994 May-Jun;5(4):165-169, Trends in endocrinology & metabolism
The Drosophila Son of sevenless (Sos) gene functions in the signaling pathway initiated by the Sevenless receptor tyrosine kinase. It encodes the Drosophila homologue of CDC25, an activator of Ras in the yeast Saccharomyces cerevisiae. Two widely expressed mammalian homologues of Sos (mSos) have now been identified and characterized. They encode for 150-kD proteins that are Ras-specific guanine nucleotide exchange factors. Genetic and biochemical studies indicate that Sos proteins bind directly to the SH2- and SH3-domain-containing adaptor protein GRB2/Drk. This interaction defines a pathway by which receptor tyrosine kinases can communicate with Ras
— id: 105531, year: 1994, vol: 5, page: 165, stat: Journal Article,

Crosslinking of the surface immunoglobulin receptor in B lymphocytes induces a redistribution of neurofibromin but not p120-GAP
Boyer MJ; Gutmann DH; Collins FS; Bar-Sagi D
1994 Feb;9(2):349-357, Oncogene
The activation of Ras proteins is a key step in the signal transduction pathways triggered by ligand-bound cell surface receptors. The GTPase activating proteins (GAPs) p120-GAP and neurofibromin, the neurofibromatosis-type 1 (NF1) gene product, are thought to play an essential role in the regulation of Ras activity by increasing the GTPase activity of wild type, but not activated Ras in vitro. Both GAPs are widely expressed in mammalian tissues thus raising the question of whether or not they have different regulatory functions. In this study, we have analysed the distribution of p120-GAP and neurofibromin in splenic B lymphocytes by immunofluorescent staining. Crosslinking of surface immunoglobulin (slg), the B-lymphocyte antigen receptor, induced the redistribution of neurofibromin. In contrast, no apparent change in the cellular localization of p120-GAP occurred followed the cross-linking of slg. The redistribution of neurofibromin coincided both spatially and temporally with the relocalization of crosslinked slg and was inhibited by the cytoskeletal disrupting agents colchicine and cytochalasin D. These findings indicated that neurofibromin and p120-GAP can be differentially regulated in vivo and suggest that neurofibromin is a component of the signaling pathway initiated by crosslinking of B lymphocyte slg. Furthermore, our observations that cocapping neurofibromin with slg is independent of the p21ras redistribution suggests that the role of neurofibromin in B cells is not solely related to its ability to act as a Ras regulator
— id: 62981, year: 1994, vol: 9, page: 349, stat: Journal Article,

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane
Rotin D; Bar-Sagi D; O'Brodovich H; Merilainen J; Lehto VP; Canessa CM; Rossier BC; Downey GP
1994 Oct 3;13(19):4440-4450, EMBO journal
The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells
— id: 62980, year: 1994, vol: 13, page: 4440, stat: Journal Article,

Activation of Ras and other signaling pathways by receptor tyrosine kinases
Schlessinger J; Bar-Sagi D
1994 ;59:173-179, Cold Spring Harbor symposia on quantitative biology
— id: 56846, year: 1994, vol: 59, page: 173, stat: Journal Article,

Inhibition of Ras-induced DNA synthesis by expression of the phosphatase MKP-1
Sun H; Tonks NK; Bar-Sagi D
1994 Oct 14;266(5183):285-288, Science
Mitogen-activated protein kinases (MAP kinases) are common components of signaling pathways induced by diverse growth stimuli. Although the guanidine nucleotide-binding Ras proteins are known to be upstream activators of MAP kinases, the extent to which MAP kinases directly contribute to the mitogenic effect of Ras is as yet undefined. In this study, inhibition of MAP kinases by the MAP kinase phosphatase MKP-1 blocked the induction of DNA synthesis in quiescent rat embryonic fibroblast REF-52 cells by an activated mutant of Ras, V12Ras. These results suggest an essential role for activation of MAP kinases in the transition from the quiescent to the DNA replication phase of the eukaryotic cell cycle
— id: 62979, year: 1994, vol: 266, page: 285, stat: Journal Article,

SH3 domains direct cellular localization of signaling molecules
Bar-Sagi D; Rotin D; Batzer A; Mandiyan V; Schlessinger J
1993 Jul 16;74(1):83-91, Cell
In this study we describe the cellular distribution of the SH2 and SH3 domains of phospholipase C-gamma (PLC-gamma) and of the adaptor protein GRB2 following their microinjection into living rat embryo fibroblasts. Using immunofluorescence microscopy, we show that a truncated protein composed of the SH2 and SH3 domains of PLC-gamma was localized to the actin cytoskeleton. A similar localization pattern was observed when only the SH3 domain of PLC-gamma was microinjected. In contrast, a truncated protein composed of only the SH2 domains of PLC-gamma exhibited diffuse cytoplasmic distribution. Microinjected GRB2 protein was localized primarily to membrane ruffles, as was GRB2 protein containing SH2 loss-of-function point mutations. Hence, the localization of GRB2 to membrane ruffles does not require interaction with tyrosine-phosphorylated moieties. However, GRB2 proteins with SH3 loss-of-function point mutations exhibited diffuse cytoplasmic distribution. These results indicate that SH3 domains are responsible for the targeting of signaling molecules to specific subcellular locations
— id: 35758, year: 1993, vol: 74, page: 83, stat: Journal Article,

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2
Chardin P; Camonis JH; Gale NW; van Aelst L; Schlessinger J; Wigler MH; Bar-Sagi D
1993 May 28;260(5112):1338-1343, Science
A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1
— id: 62984, year: 1993, vol: 260, page: 1338, stat: Journal Article,

Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras
Gale NW; Kaplan S; Lowenstein EJ; Schlessinger J; Bar-Sagi D
1993 May 6;363(6424):88-92, Nature
Activation of receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, or nerve growth factor converts the inactive, GDP-bound form of Ras to the active, GTP-bound form, and a dominant negative mutant of Ras interferes with signalling from such receptors. The mechanisms by which receptor tyrosine kinases and Ras are coupled, however, are not well understood. Many cytoplasmic proteins regulated by such receptors contain Src-homology (SH) 2 and 3 domains, and the SH2- and SH3-containing protein Grb2, like its homologue from Caenorhabditis elegans, Sem-5, appears to play an important role in the control of Ras by receptor tyrosine kinases. Here we show that overexpression of Grb2 potentiates the EGF-induced activation of Ras and mitogen-activated protein kinase by enhancing the rate of guanine nucleotide exchange on Ras. Cellular Grb2 appears to form a complex with a guanine-nucleotide-exchange factor for Ras, which binds to the ligand-activated EGF receptor, allowing the tyrosine kinase to modulate Ras activity
— id: 62985, year: 1993, vol: 363, page: 88, stat: Journal Article,

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event
Hide I; Bennett JP; Pizzey A; Boonen G; Bar-Sagi D; Gomperts BD; Tatham PE
1993 Nov;123(3):585-593, Journal of cell biology
Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells
— id: 62982, year: 1993, vol: 123, page: 585, stat: Journal Article,

Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling
Li N; Batzer A; Daly R; Yajnik V; Skolnik E; Chardin P; Bar-Sagi D; Margolis B; Schlessinger J
1993 May 6;363(6424):85-88, Nature
Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling
— id: 57448, year: 1993, vol: 363, page: 85, stat: Journal Article,

Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts
Quelle DE; Ashmun RA; Shurtleff SA; Kato JY; Bar-Sagi D; Roussel MF; Sherr CJ
1993 Aug;7(8):1559-1571, Genes & development
Mammalian D-type cyclins are growth factor-regulated, delayed early response genes that are presumed to control progression through the G1 phase of the cell cycle by governing the activity of cyclin-dependent kinases (cdks). Overexpression of mouse cyclin D1 in serum-stimulated mouse NIH-3T3 and rat-2 fibroblasts increased their rates of G0 to S- and G1- to S-phase transit by several hours, leading to an equivalent contraction of their mean cell generation times. Although such cells remained contact inhibited and anchorage dependent, they manifested a reduced serum requirement for growth and were smaller in size than their normal counterparts. Ectopic expression of cyclin D2 in rodent fibroblasts, either alone or together with exogenous cdk4, shortened their G0- to S-phase interval and reduced their serum dependency, but cyclin D2 alone did not alter cell size significantly. When cells were microinjected during the G1 interval with a monoclonal antibody specifically reactive to cyclin D1, parental rodent fibroblasts and derivatives overexpressing this cyclin were inhibited from entering S phase, but cells injected near the G1/S phase transition were refractory to antibody-induced growth suppression. Thus, cyclin D1, and most likely D2, are rate limiting for G1 progression
— id: 62983, year: 1993, vol: 7, page: 1559, stat: Journal Article,

Mechanisms of signal transduction by Ras
Bar-Sagi D
1992 Apr;3(2):93-98, Seminars in cell biology
The role of Ras in the transduction of signals that control cell growth is undisputed. However, the identity of the Ras signalling pathway remains unknown. Evidence is mounting that Ras can receive signals from different cell surface receptors most likely via a common intermediate, GAP. A new insight into the possible function of Ras is provided by the recent findings that certain ligands can induce the coordinated redistribution of Ras and cell surface receptors. The next challenge is to identify the specific targets for the action of Ras
— id: 62986, year: 1992, vol: 3, page: 93, stat: Journal Article,

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling
Lowenstein EJ; Daly RJ; Batzer AG; Li W; Margolis B; Lammers R; Ullrich A; Skolnik EY; Bar-Sagi D; Schlessinger J
1992 Aug 7;70(3):431-442, Cell
A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling
— id: 13480, year: 1992, vol: 70, page: 431, stat: Journal Article,

Anoxia induces phospholipase A2 activation in rabbit renal proximal tubules
Portilla D; Mandel LJ; Bar-Sagi D; Millington DS
1992 Mar;262(3 Pt 2):F354-F360, American journal of physiology
Phospholipase A2 (PLA2) activation during anoxic cell injury was determined by use of a variety of approaches in rabbit proximal renal tubules. Arachidonic acid (AA) mass release increased from 4 +/- 1 (normoxia control) to 40 +/- 6 ng/mg protein after 20 min and 106 +/- 16 ng/mg protein after 40 min of anoxia. PLA2 activity was measured by estimating the amount of sn-2 fatty acid released from either 14C-labeled Escherichia coli membranes or [14C]phosphatidylethanolamine (PE) micelles incubated with membrane and cytosolic fractions obtained from normoxic or anoxic tubules. At pH 7.4 and 1 mM Ca, PLA2 activity increased in the 20-min anoxic membrane fractions from 8.1 +/- 2.3 (normoxic) to 15.2 +/- 2.1 pmol.min-1.mg protein-1 (anoxic). When the proximal tubules were homogenized in the absence of Ca, the anoxia-induced PLA2 activity was found to be soluble. Preincubation with pancreatic PLA2 antibody inhibited 50% of both basal and anoxia-stimulated PLA2 activity. Two protein bands (40- and 21-kDa species) immunoreactive to PLA2 antibody were detected in the membrane fraction. A sixfold increase in the immunoreactivity of the 40-kDa band was detected after 40 min of anoxia of proximal tubules. These results suggest that anoxia induces an intracellular PLA2 activity in kidney cells that could be immunologically related to pancreatic PLA2
— id: 62987, year: 1992, vol: 262, page: F354, stat: Journal Article,

Phospholipase A2: microinjection and cell localization techniques
Bar-Sagi D
1991 ;197:269-279, Methods in enzymology
— id: 62990, year: 1991, vol: 197, page: 269, stat: Journal Article,

Introduction of unlabeled proteins into living cells by electroporation and isolation of viable protein-loaded cells using dextran-fluorescein isothiocyanate as a marker for protein uptake
Graziadei L; Burfeind P; Bar-Sagi D
1991 Apr;194(1):198-203, Analytical biochemistry
Commonly, microinjection has been the method of choice for introducing proteins into living cells. Viable cells containing an introduced protein can be then identified providing that the protein is fluorochrome conjugated. This approach is applicable only for adherent cells, and the number of cells that can be analyzed is small. In this study, we have established that electroporation can be used to load proteins into large numbers of cells with high efficiency. Furthermore, we have developed a method for the isolation of protein-loaded cells using fluorescein isothiocyanate-dextran (dextran-FITC) as a molecular marker for protein uptake. The essential features of this method are that dextran-FITC is included in the electroporation medium and, thus, is cointroduced with the protein of interest. Purification of cells containing dextran-FITC using fluorescence-activated cell sorting yields a population which is composed almost entirely of cells containing the protein of interest
— id: 62989, year: 1991, vol: 194, page: 198, stat: Journal Article,

Association of p21ras with cellular polypeptides
Kaplan S; Bar-Sagi D
1991 Oct 5;266(28):18934-18941, Journal of biological chemistry
p21ras specific antiserum was used to immunoprecipitate p21ras polypeptides from human A431 cells. In addition to p21ras, this antiserum precipitated a series of polypeptides with relative molecular weights of 150,000, 120,000, 105,000, and 50,000. The precipitation of these polypeptides was prevented by preincubation of the antiserum with an excess of purified Ras protein. These polypeptides do not share an epitope with p21ras, and two of them (120 and 150 kDa) copurify with a fraction of p21ras. The co-precipitation of p21ras with these polypeptides was detected in a variety of cell types. The pattern of the immunoprecipitates was consistently different in normal and ras-transformed cells. The 120- and 150-kDa polypeptides are phosphorylated on serine and threonine in A431 cells. Serum treatment resulted in a 2-fold increase in the phosphoserine content of the 120-kDa polypeptides
— id: 62988, year: 1991, vol: 266, page: 18934, stat: Journal Article,

Effect of H-ras proteins on the activity of polyphosphoinositide phospholipase C in HL60 membranes
Cockcroft S; Bar-Sagi D
1990 ;2(3):227-234, Cellular signalling
The present study was undertaken to investigate whether purified ras proteins can affect the activity of polyphosphoinositide specific phospholipase C in a cell-free membrane system. For this purpose we used homogenous preparations of the proto-oncogenic (H-ras(gly 12)) and the oncogenic (H-ras(val 12)) forms of the human H-ras proteins and membranes prepared from the human leukemic HL60 cells. We demonstrate that both the proto-oncogenic and the oncogenic form of H-ras proteins stimulate phospholipase C activity only when coupled to non-hydrolysable analogues of GTP
— id: 62992, year: 1990, vol: 2, page: 227, stat: Journal Article,

Co-capping of ras proteins with surface immunoglobulins in B lymphocytes
Graziadei L; Riabowol K; Bar-Sagi D
1990 Sep 27;347(6291):396-400, Nature
Cellular ras genes encode a family of membrane-associated proteins (p21ras) that bind guanine nucleotide and possess a low intrinsic GTPase activity. The p21ras proteins are ubiquitously expressed in mammalian cells and are thought to be involved in a growth-promoting signal transduction pathway; their mode of action, however, remains unknown. The ligand-induced movement of cell-surface receptors seems to be a primary event in the transduction of several extracellular signals that control cell growth and differentiation. In B lymphocytes, surface immunoglobulin receptors crosslinked by antibody or other multivalent ligands form aggregates called patches, which then collect into a single assembly, a cap, at one pole of the cell. This process constitutes the initial signal for the activation of a B cell. Here we show by immunofluorescence microscopy that p21ras co-caps with surface immunoglobulin molecules in mouse splenic B lymphocytes. In contrast, no apparent change in the distribution of p21ras occurs during the capping of concanavalin A receptors. The redistribution of p21ras is apparent at the early stages (patching) of immunoglobulin capping and is inhibited by metabolic inhibitors and the cytoskeleton-disrupting agents colchicine and cytochalasin D. The distribution of another membrane-associated guanine nucleotide-binding regulatory protein, the Gi alpha subunit, is not affected by surface immunoglobulin capping. These findings demonstrate that p21ras can migrate in a directed manner along the plasma membrane and suggest that p21ras may be a component of the signalling pathway initiated by the capping of surface immunoglobulin in B lymphocytes
— id: 62991, year: 1990, vol: 347, page: 396, stat: Journal Article,

ras proteins: biological effects and biochemical targets (review)
Bar-Sagi D
1989 Sep-Oct;9(5):1427-1437, Anticancer research
Ras genes are an ubiquitous eukaryotic gene family. Since their discovery as the cellular homologues of the transforming genes of Harvey and Kirsten retroviruses, ras genes have been presumed to play a role in growth control, mainly because of their potential to induce uncontrolled cell proliferation. This notion is strongly supported by recent evidence indicating that ras mutations may be causative or closely linked to the onset of some types of human tumors. However, the mechanism of action of ras proteins in mammalian cells is poorly understood. Using the microinjection technique as a biological assay for ras proteins, it has been possible to address several important questions concerning cellular and biochemical aspects of ras function. When introduced into living cells by microinjection, purified ras proteins can induce cell proliferation, neuronal differentiation, oocyte maturation, and exocytotic degranulation. On the biochemical level, microinjection studies indicated that ras proteins can induce specific alterations in phospholipid metabolism
— id: 62993, year: 1989, vol: 9, page: 1427, stat: Journal Article,

G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2
Kim D; Lewis DL; Graziadei L; Neer EJ; Bar-Sagi D; Clapham DE
1989 Feb 9;337(6207):557-560, Nature
Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers
— id: 62994, year: 1989, vol: 337, page: 557, stat: Journal Article,

Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells
Bar-Sagi D; Gomperts BD
1988 Oct;3(4):463-469, Oncogene
To investigate the possible role of ras proteins in the secretory process, we have microinjected the proto oncogenic and oncogenic forms of the human H-ras protein into rat peritoneal mast cells. Mast cells are secretory cells which, upon appropriate stimulus, liberate histamine and other mediators of the acute inflammatory reaction by exocytotic degranulation. We report here that microinjection of the ras oncogene protein into mast cells induces exocytotic degranulation. In contrast, microinjection of similar amounts of the proto-oncogenic protein has little apparent effect on mast cells. Degranulation induced by injection of the ras oncogene protein occurs in the absence of an external stimulus and requires the presence of external calcium. The ultrastructural features of exocytotic degranulation in mast cells injected with the ras oncogene protein are similar to those seen when mast cells are activated by soluble ligands. Our results suggest that ras proteins may be involved, possibly as regulatory elements, in cellular functions that control exocytosis
— id: 62995, year: 1988, vol: 3, page: 463, stat: Journal Article,

Localization of phospholipase A2 in normal and ras-transformed cells
Bar-Sagi D; Suhan JP; McCormick F; Feramisco JR
1988 May;106(5):1649-1658, Journal of cell biology
The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed
— id: 62996, year: 1988, vol: 106, page: 1649, stat: Journal Article,

Regulation of membrane turnover by ras proteins
Bar-Sagi D; Fernandez A; Feramisco JR
1987 May;7(5):427-434, Bioscience reports
Because ras oncogenes mediate abnormal cellular growth, ras proteins have been presumed to play a role primarily in growth control. The biological function of ras proteins may, however, prove to be much more diverse: ras proteins may be involved in cellular functions that control endocytosis and/or exocytosis
— id: 62997, year: 1987, vol: 7, page: 427, stat: Journal Article,

Inhibition of cell surface ruffling and fluid-phase pinocytosis by microinjection of anti-ras antibodies into living cells
Bar-Sagi D; McCormick F; Milley RJ; Feramisco JR
1987 ;Suppl 5:69-73, Journal of cellular physiology. Supplement
Fibroblasts transformed by ras oncogenes display enhanced cell surface ruffling and fluid-phase pinocytotic activities. Microinjection of antibodies that specifically bind the ras proteins into these cells results in the inhibition of these two surface activities. The possible underlying biochemical basis of the influence of the ras proteins on membrane ruffling and pinocytosis and the potential relationship of these two biological activities to membrane signal transduction are discussed
— id: 62998, year: 1987, vol: Suppl 5, page: 69, stat: Journal Article,

Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins
Bar-Sagi D; Feramisco JR
1986 Sep 5;233(4768):1061-1068, Science
Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins
— id: 62999, year: 1986, vol: 233, page: 1061, stat: Journal Article,

Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation
Bar-Sagi D; Feramisco JR
1985 Oct;42(3):841-848, Cell
To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein
— id: 63000, year: 1985, vol: 42, page: 841, stat: Journal Article,

Negative modulation of sodium channels in cultured chick muscle cells by the channel activator batrachotoxin
Bar-Sagi D; Prives J
1985 Apr 25;260(8):4740-4744, Journal of biological chemistry
We have investigated the possibility that cellular control of membrane excitability involves feedback mechanisms in which the degree of activity of voltage-sensitive Na+ channels regulates the number of these channels. Using two independent assays, channel-mediated Na+ uptake and the specific binding of [3H] saxitoxin, we have studied the effects of pharmacological activation of Na+ channels with batrachotoxin (BTX) on the number and properties of these channels. Upon exposure of cultured muscle cells to BTX (1 microM), the number of surface Na+ channels decreases by approximately 75%, with a half-time of 3-6 h. This decrease is prevented by pharmacological blockade of these channels and does not reflect changes in the apparent affinities towards either BTX or saxitoxin. This reduction is reversible: a gradual increase in surface Na+ channels that is dependent on protein synthesis is observed upon removal of the activator. The BTX-induced decrease in Na+ channels is associated with an enhanced rate of disappearance of surface Na+ channels. These findings point to the existence of a down-regulation mechanism for the modulation of membrane excitability under conditions of elevated Na+ channel activity
— id: 63001, year: 1985, vol: 260, page: 4740, stat: Journal Article,

Regulation of surface properties in cultured muscle cells
Bar-Sagi, Dafna
[S.l. : s.n.], 1984,
Thesis (Ph.D.) -- State University of New York at Stony Brook, 1984
— id: 2082, year: 1984, vol: , page: , stat: ,

Trifluoperazine, a calmodulin antagonist, inhibits muscle cell fusion
Bar-Sagi D; Prives J
1983 Nov;97(5 Pt 1):1375-1380, Journal of cell biology
We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion
— id: 63002, year: 1983, vol: 97, page: 1375, stat: Journal Article,

Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells
Bar-Sagi D; Prives J
1983 Jan;114(1):77-81, Journal of cellular physiology
We have investigated the effect of tunicamycin (TM), an inhibitor of protein glycosylation, on surface Na+ channels in cultured chick skeletal muscle cells. The expression of Na+ channels, estimated by the measurement of batrachotoxin (BTX)-activated 22Na+ uptake, was found to be significantly diminished after exposure of muscle cells to TM. This effect is partially reversed by the protease inhibitor leupeptin and is associated with a markedly enhanced rate of disappearance of Na+ channels from the surface of TM-treated cells. Our findings suggest that protein glycosylation contributes to the metabolic stability of voltage-sensitive Na+ channels
— id: 63005, year: 1983, vol: 114, page: 77, stat: Journal Article,

Effect of tunicamycin, an inhibitor of protein glycosylation, on the biological properties of acetylcholine receptor in cultured muscle cells
Prives J; Bar-Sagi D
1983 Feb 10;258(3):1775-1780, Journal of biological chemistry
We have studied the effect of tunicamycin (TM), an antibiotic which inhibits the glycosylation of nascent proteins, on the properties of the acetylcholine receptor (AChR) at the surface of embryonic chick skeletal muscle cells. The use of two separate assays, specific binding of 125I-alpha-bungarotoxin and carbamylcholine-activated 22Na+ uptake, has allowed us to monitor the effects of impaired glycosylation on the metabolic and functional properties of AChR. A significant decrease in the amounts of surface AChR elaborated in the presence of TM is detected by both measurements. This decrease has been found to reflect an enhanced proteolytic degradation of the underglycosylated AChR. The underglycosylated AChR, expressed on the cell surface in the presence of TM, retains the capability of mediating agonist-activated ionic permeability changes, but displays quantitatively altered interactions with receptor ligands. We conclude that the carbohydrate moiety on AChR may play a role in determining the folding of newly synthesized polypeptides to form a conformation compatible with the metabolic properties and ligand interactions characteristic of glycosylated AChR
— id: 63004, year: 1983, vol: 258, page: 1775, stat: Journal Article,

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture
Strichartz G; Bar-Sagi D; Prives J
1983 Sep;82(3):365-384, Journal of general physiology
The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells
— id: 63003, year: 1983, vol: 82, page: 365, stat: Journal Article,