DOCSLIB.ORG

Rasgrp1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression During Multistage Skin Carcinogenesis Courtney T

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Rasgrp1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression During Multistage Skin Carcinogenesis Courtney T Research Article RasGRP1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression during Multistage Skin Carcinogenesis Courtney T. Luke,1 Carolyn E. Oki-Idouchi,1 J. Mark Cline,2 and Patricia S. Lorenzo1 1Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii and 2Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina Abstract diacylglycerol and its ultrapotent analogues, the phorbol esters (3). RasGRP1 is a guanine nucleotide exchange factor for Ras, Previous studies on RasGRP1 and RasGRP3 showed their high- activated in response to the second messenger diacylglycerol affinity binding to diacylglycerol analogues (4–6), resulting in the and its ultrapotent analogues, the phorbol esters. We have activation of Ras and Ras signaling cascades, and suggesting that previously shown that RasGRP1 is expressed in mouse pathways besides PKC could transmit signals from diacylglycerol to epidermal keratinocytes and that transgenic mice over- Ras. In fact, the discovery of RasGRP1 has led to a better under- expressing RasGRP1 in the epidermis under the keratin 5 standing of the link between T-cell receptor stimulation and phos- promoter (K5.RasGRP1) are prone to developing spontaneous pholipase C activation with Ras signaling (7). In contrast to PKC, RasGRP members have limited tissue distribution. RasGRP1, for papillomas and squamous cell carcinomas, suggesting a role for RasGRP1 in skin tumorigenesis. Here, we examined the example, is expressed in Tcells and at lower levels in B cells, neurons, response of the K5.RasGRP1 mice to multistage skin carcino- mastocytes, and some kidney cells (8–12). Recently, we have also genesis, using 7,12-dimethylbenz(a)anthracene as carcinogen found RasGRP1 expression in epidermal keratinocytes, where it can and 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor mediate Ras activation in response to phorbol esters (13, 14). promoter. We found that whereas tumor multiplicity did not The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) differ between transgenic and wild-type groups, the transgenic was first described as a potent skin tumor promoter and later tumors were significantly larger than those observed in the identified as a diacylglycerol mimetic able to bind to PKC (15). Thus, wild-type mice (wild-type, 4.58 F 0.25mm; transgenic, 9.83 F TPA-induced skin tumor promotion has been primarily linked to 1.05mm). Histologic analysis further revealed that squamous the modulation of PKC in keratinocytes, although the contribution cell carcinomas generated in the transgenic mice were less of the individual PKC isoforms to this effect is complex and still not differentiated and more invasive than the wild-type tumors. fully understood (16). In addition to the role of PKC, differences in Additionally, 30% of the transgenic mice developed tumors in susceptibility to tumor promotion among mouse strains suggest the absence of initiation, suggesting that RasGRP1 over- that there are modifier genes that can influence the response of the expression could partially substitute for the initiation step epidermis to the phorbol esters. Analysis of traits associated with induced by dimethylbenz(a)anthracene. In primary keratino- increased or decreased tumor promotion susceptibility revealed cytes isolated from K5.RasGRP1 mice, TPA stimulation several genetic loci, including one locus in mouse chromosome 2 induced higher levels of Ras activation compared with the (17). Nmes1, whose role and relationshipwith TPA signaling is levels measured in the wild-type cells, indicating that unknown, is one of the genes identified in chromosome 2 that was constitutive overexpression of RasGRP1 in epidermal cells found elevated in the skin of susceptible mouse strains (18). leads to elevated biochemical activation of endogenous Ras in Interestingly, RasGRP1 also maps near the TPA promotion response to TPA. The present data suggests that RasGRP1 susceptibility locus in chromosome 2 (17), although its participation participates in skin carcinogenesis via biochemical activation in tumor promotion by TPA has not been explored. To investigate of endogenous wild-type Ras and predisposes to malignant the potential role of RasGRP1 in skin tumorigenesis, we recently progression in cooperation with Ras oncogenic signals. developed a transgenic mouse model for the overexpression of [Cancer Res 2007;67(21):10190–7] RasGRP1 in the epidermis (K5.RasGRP1; ref. 19). These mice are prone to developing spontaneous papillomas and squamous cell Introduction carcinomas (SCCs), implying a role for RasGRP1 in tumor initiation (19). To gain further insight into the effect of RasGRP1 during skin RasGRP is a family of guanine nucleotide exchange factors for carcinogenesis, particularly TPA-induced tumor promotion, we Ras/Rapsmall GTPases and is composedof four members subjected K5.RasGRP1 mice to the classic two-stage chemical (RasGRP1–4) which differ in their substrate recognition (1, 2). All carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene the RasGRP members possess a cysteine-rich domain that shares (DMBA) as an initiator. We found that RasGRP1 overexpression in similar features as the C1 domain motif of protein kinase C (PKC), skin did not affect tumor formation in response to DMBA/TPA, but which is responsible for binding to the second messenger caused larger and more invasive tumors than those observed in the wild-type animals. These findings indicate that RasGRP1 modula- tion was dispensable for the TPA-induced tumor promotion of Note: C.T. Luke and C.E. Oki-Idouchi contributed equally to this work. Requests for reprints: Patricia S. Lorenzo, Cancer Research Center of Hawaii, 651 carcinogen-initiated skin, but contributed to tumor progression, Ilalo Street, Room 222-K, Honolulu, HI 96813. Phone: 808-586-5868; Fax: 808-587-0742; suggesting that elevated biochemical activation of Ras through E-mail: [email protected]. I2007 American Association for Cancer Research. increased expression of RasGRP1 could cooperate with Ras doi:10.1158/0008-5472.CAN-07-2375 oncogenic signals towards malignancy. Cancer Res 2007; 67: (21). November 1, 2007 10190 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. RasGRP1 in Multistage Skin Carcinogenesis Materials and Methods CDD camera (Roper Scientific) at Â400 magnification and the thickness of the epidermis was measured in micrometers using MetaMorph (Molecular Animals and skin carcinogenesis experiments. The K5.RasGRP1 mice Devices Corporation). The grid of a hemacytometer was used for calibration were generated as previously described on the FVB/N background strain of the MetaMorph software. Each sample was measured at three different (19). For the skin carcinogenesis experiments, the back of 6- to 8-week-old locations before calculating the average thickness within each treatment mice, both males and females, were shaved with electric clippers 2 days group. before the beginning of the protocol. The mice in each group, transgenic Histopathology and immunohistochemistry. Skin tumors were fixed and wild-type, were divided into two cohorts of 16 animals each of mixed in 4% paraformaldehyde for 24 h and maintained in 70% ethanol until gender to be treated with either DMBA plus TPA or acetone plus TPA. The paraffin-embedded. H&E-stained slides were used for descriptive histopa- A DMBA treatment consisted of a single topical application of 26 gofDMBA thology. Immunohistochemical localization of the transgenic RasGRP1-HA A in 200 L of acetone on the shaved dorsal skin; TPA treatment was initiated protein was performed as previously described (19). Briefly, deparaffinized A 2 weeks after DMBA initiation by topical application of 2 g of TPA in 200 sections were subjected to heat-induced epitope retrieval. After blocking, AL of acetone twice a week for 20 weeks. Animals were monitored at the tissues were incubated with anti-HA antibody (Santa Cruz Biotechnology) time of TPA applications, and tumors were counted and measured with a followed by HRP-conjugated AffinityPure donkey anti-rabbit F(ab¶)2 caliper at least once a week. After the TPA treatment was ended, mice were fragment–specific antibody (Jackson ImmunoResearch). 3,3¶-Diaminoben- followed for an additional 8 weeks and then euthanized by CO2 zidine was used as a substrate (Dako). Tissues were counterstained with asphyxiation. Tumor samples were collected and fixed for histology. Mayer’s hematoxylin (InnoGenex). Epidermal hyperplasia. TPA-induced acute hyperplasia was evaluated RasGTP pull-down assay and Western blots. Levels of GTP-loaded Ras in both K5.RasGRP1 and wild-type mice of 6 to 8 weeks of age after 48 (RasGTP) were measured by using the GST-RBD domain of Raf-1 as a probe h treatment with 3 Ag of TPA in 200 AL of acetone applied to the dorsal skin. in an affinity precipitation or pull-down assay. Briefly, primary keratinocytes The skin area to be treated was shaved with electric clippers 2 days before were first isolated from K5.RasGRP1 or wild-type mice as described TPA treatment. Control, acetone treatments, were also done in place of TPA. elsewhere (14). Cells were serum starved overnight, treated with vehicle A The epidermal thickness was determined by microscopic examination of (Me2SO) or 1 mol/L of TPA for 15 min, and harvested on ice in lysis buffer H&E-stained skin samples. Microphotographs were taken with a CoolSnap containing 25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L of NaCl, Figure 1. Skin tumor development in K5.RasGRP1 transgenic mice subjected to two-stage carcinogenesis. A, tumor multiplicity (average number oftumors per mouse F SE) and (B) incidence (percentage ofmice with tumors) in wild-type ( o) and K5.RasGRP1 transgenic (.) mice treated with TPA following initiation with DMBA. C, wild-type (Wt) and K5.RasGRP1 transgenic (Tg) mice bearing tumors. Pictures were taken at the end ofthe protocol. D, tumor size (diameter in millimeters) at 17 and 28 wk after initiation with DMBA in both wild-type (Wt) and K5.RasGRP1 transgenic (Tg) mice.
Load more

Recommended publications
  • Genetic and Genomic Analysis of Hyperlipidemia, Obesity and Diabetes Using (C57BL/6J × TALLYHO/Jngj) F2 Mice
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Nutrition Publications and Other Works Nutrition 12-19-2010 Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P. Stewart Marshall University Hyoung Y. Kim University of Tennessee - Knoxville, [email protected] Arnold M. Saxton University of Tennessee - Knoxville, [email protected] Jung H. Kim Marshall University Follow this and additional works at: https://trace.tennessee.edu/utk_nutrpubs Part of the Animal Sciences Commons, and the Nutrition Commons Recommended Citation BMC Genomics 2010, 11:713 doi:10.1186/1471-2164-11-713 This Article is brought to you for free and open access by the Nutrition at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Nutrition Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Stewart et al. BMC Genomics 2010, 11:713 http://www.biomedcentral.com/1471-2164/11/713 RESEARCH ARTICLE Open Access Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P Stewart1, Hyoung Yon Kim2, Arnold M Saxton3, Jung Han Kim1* Abstract Background: Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/ JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.
    [Show full text]
  • Mechanistic Characterization of RASGRP1 Variants Identifies an Hnrnp K-Regulated Transcriptional Enhancer Contributing to SLE Susceptibility
    bioRxiv preprint doi: https://doi.org/10.1101/568790; this version posted March 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Mechanistic characterization of RASGRP1 variants identifies an hnRNP K-regulated transcriptional enhancer contributing to SLE susceptibility Julio E. Molineros1*, Bhupinder Singh1*, Chikashi Terao2,3, Yukinori Okada4, Jakub Kaplan1, Barbara McDaniel1, Shuji Akizuki3, Celi Sun1, Carol Webb5, Loren L. Looger6, Swapan K. Nath1 1Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 2Laboratory for Statistical Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan. 3Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan. 4Department of Statistical Genetics, Osaka University Graduate School of Medicine, Osaka, Japan 5Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA 6Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA *Contributed equally Corresponding author Swapan K. Nath, Ph.D. Member Arthritis & Clinical Immunology Program Oklahoma Medical Research Foundation 825 NE 13th St. Oklahoma City, OK 73104 Ph: (405)-271-7765 Fax: (405)-271-4110 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/568790; this version posted March 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Patients with Systemic Lupus Erythematosus Nucleotide-Releasing Protein 1 in a Subset of Defective Expression of Ras Guanyl
    Defective Expression of Ras Guanyl Nucleotide-Releasing Protein 1 in a Subset of Patients with Systemic Lupus Erythematosus This information is current as Shinsuke Yasuda, Richard L. Stevens, Tomoko Terada, of September 24, 2021. Masumi Takeda, Toko Hashimoto, Jun Fukae, Tetsuya Horita, Hiroshi Kataoka, Tatsuya Atsumi and Takao Koike J Immunol 2007; 179:4890-4900; ; doi: 10.4049/jimmunol.179.7.4890 http://www.jimmunol.org/content/179/7/4890 Downloaded from References This article cites 39 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/179/7/4890.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Defective Expression of Ras Guanyl Nucleotide-Releasing Protein 1 in a Subset of Patients with Systemic Lupus Erythematosus1 Shinsuke Yasuda,2* Richard L.
    [Show full text]
  • Immune Adaptor SKAP1 Acts a Scaffold for Polo-Like Kinase 1 (PLK1)
    www.nature.com/scientificreports OPEN Immune adaptor SKAP1 acts a scafold for Polo-like kinase 1 (PLK1) for the optimal cell cycling Received: 14 September 2018 Accepted: 6 June 2019 of T-cells Published: xx xx xxxx Monika Raab1,2, Klaus Strebhardt1 & Christopher E. Rudd2,3,4 While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock- down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our fndings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation. Polo-like kinase 1 (PLK1) is a serine/threonine kinase that regulates the mitosis of mammalian cells. It is a mem- ber of the Polo-like serine/threonine kinase (PLKs) family that are structurally conserved from yeast to mam- mals.
    [Show full text]
  • Structural Analysis of Autoinhibition in the Ras-Specific Exchange Factor
    RESEARCH ARTICLE elife.elifesciences.org Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1 Jeffrey S Iwig1,2, Yvonne Vercoulen3†, Rahul Das1,2†, Tiago Barros1,2,4, Andre Limnander3, Yan Che1,2, Jeffrey G Pelton2, David E Wemmer2,5,6, Jeroen P Roose3*, John Kuriyan1,2,4,5,6* 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; 2California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States; 3Department of Anatomy, University of California, San Francisco, San Francisco, United States; 4Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; 5Department of Chemistry, University of California, Berkeley, Berkeley, United States; 6Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States Abstract RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals.
    [Show full text]
  • Global Transcriptome Analysis Identifies Weight Regain-Induced
    OPEN International Journal of Obesity (2018) 42, 755–764 www.nature.com/ijo ORIGINAL ARTICLE Global transcriptome analysis identifies weight regain-induced activation of adaptive immune responses in white adipose tissue of mice DS Kyung1,2,3,7, HR Sung1,2,7, YJ Kim1,2, KD Kim4, SY Cho2,5, JH Choi4, Y-H Lee6, IY Kim1,2 and JK Seong1,2,3 OBJECTIVE: Studies have indicated that weight regain following weight loss predisposes obese individuals to metabolic disorders; however, the molecular mechanism of this potential adverse effect of weight regain is not fully understood. Here we investigated global transcriptome changes and the immune response in mouse white adipose tissue caused by weight regain. DESIGN: We established a diet switch protocol to compare the effects of weight regain with those of weight gain without precedent weight loss, weight loss maintenance and chow diet. We conducted a time course analysis of global transcriptome changes in gonadal white adipose tissue (gWAT) during the weight fluctuation. Co-expression network analysis was used to identify functional modules associated with the weigh regain phenotype. Immune cell populations in gWAT were characterized by flow- cytometric immunophenotyping. Metabolic phenotypes were monitored by histological analysis of adipose tissue and liver, and blood-chemistry and body weight/composition analyses. RESULTS: In total, 952 genes were differentially expressed in the gWAT in the weight regain vs the weight gain group. Upregulated genes were associated with immune response and leukocyte activation. Co-expression network analysis showed that genes involved in major histocompatibility complex I and II-mediated antigen presentation and T-cell activation function were upregulated.
    [Show full text]
  • Inflammatory, Regulatory, and Autophagy Co-Expression Modules
    Durocher et al. Journal of Neuroinflammation (2019) 16:56 https://doi.org/10.1186/s12974-019-1433-4 RESEARCH Open Access Inflammatory, regulatory, and autophagy co-expression modules and hub genes underlie the peripheral immune response to human intracerebral hemorrhage Marc Durocher1, Bradley P. Ander1, Glen Jickling1, Farah Hamade1, Heather Hull1, Bodie Knepp1, Da Zhi Liu1, Xinhua Zhan1, Anh Tran1, Xiyuan Cheng1, Kwan Ng1, Alan Yee1, Frank R. Sharp1 and Boryana Stamova1,2* Abstract Background: Intracerebral hemorrhage (ICH) has a high morbidity and mortality. The peripheral immune system and cross-talk between peripheral blood and brain have been implicated in the ICH immune response. Thus, we delineated the gene networks associated with human ICH in the peripheral blood transcriptome. We also compared the differentially expressed genes in blood following ICH to a prior human study of perihematomal brain tissue. Methods: We performed peripheral blood whole-transcriptome analysis of ICH and matched vascular risk factor control subjects (n = 66). Gene co-expression network analysis identified groups of co-expressed genes (modules) associated with ICH and their most interconnected genes (hubs). Mixed-effects regression identified differentially expressed genes in ICH compared to controls. Results: Of seven ICH-associated modules, six were enriched with cell-specific genes: one neutrophil module, one neutrophil plus monocyte module, one T cell module, one Natural Killer cell module, and two erythroblast modules. The neutrophil/monocyte modules were enriched in inflammatory/immune pathways; the T cell module in T cell receptor signaling genes; and the Natural Killer cell module in genes regulating alternative splicing, epigenetic, and post-translational modifications.
    [Show full text]
  • Rabbit Anti-Rasgrp1/FITC Conjugated Antibody
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-Rasgrp1/FITC Conjugated antibody SL10436R-FITC Product Name: Anti-Rasgrp1/FITC Chinese Name: FITC标记的Ras蛋白特异鸟嘌呤核苷酸释放因子1抗体 Calcium and DAG-regulated guanine nucleotide exchange factor II; CALDAG-GEFI; CALDAG-GEFII; Guanine nucleotide exchange factor, calcium and DAG regulated, Alias: Rap1A; hRasGRP1; Ras activator RasGRP; RAS guanyl nucleotide releasing protein 1; RAS guanyl releasing protein 1 (calcium and DAG-regulated); RAS guanyl releasing protein 1; RASGRP; RASGRP1; GRP1_HUMAN. Organism Species: Rabbit Clonality: Polyclonal React Species: Human,Mouse,Rat,Dog,Rabbit, ICC=1:50-200IF=1:50-200 Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 90kDa Form: Lyophilized or Liquid Concentration: 1mg/ml immunogen: KLH conjugated synthetic peptide derived from human Rasgrp1 Lsotype: IgGwww.sunlongbiotech.com Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. background: This gene is a member of a family of genes characterized by the presence of a Ras superfamily guanine nucleotide exchange factor (GEF) domain. It functions as a Product Detail: diacylglycerol (DAG)-regulated nucleotide exchange factor specifically activating Ras through the exchange of bound GDP for GTP.
    [Show full text]
  • Modeling of RAS Complexes Supports Roles in Cancer for Less Studied Partners H
    The Author(s) BMC Biophysics 2017, 10(Suppl 1):5 DOI 10.1186/s13628-017-0037-6 RESEARCH Open Access Modeling of RAS complexes supports roles in cancer for less studied partners H. Billur Engin, Daniel Carlin, Dexter Pratt and Hannah Carter* From VarI-SIG 2016: identification and annotation of genetic variants in the context of structure, function, and disease Orlando, Florida, USA. 09 July 2016 Abstract Background: RAS protein interactions have predominantly been studied in the context of the RAF and PI3kinase oncogenic pathways. Structural modeling and X-ray crystallography have demonstrated that RAS isoforms bind to canonical downstream effector proteins in these pathways using the highly conserved switch I and II regions. Other non-canonical RAS protein interactions have been experimentally identified, however it is not clear whether these proteins also interact with RAS via the switch regions. Results: To address this question we constructed a RAS isoform-specific protein-protein interaction network and predicted 3D complexes involving RAS isoforms and interaction partners to identify the most probable interaction interfaces. The resulting models correctly captured the binding interfaces for well-studied effectors, and additionally implicated residues in the allosteric and hyper-variable regions of RAS proteins as the predominant binding site for non-canonical effectors. Several partners binding to this new interface (SRC, LGALS1, RABGEF1, CALM and RARRES3) have been implicated as important regulators of oncogenic RAS signaling. We further used these models to investigate competitive binding and multi-protein complexes compatible with RAS surface occupancy and the putative effects of somatic mutations on RAS protein interactions. Conclusions: We discuss our findings in the context of RAS localization to the plasma membrane versus within the cytoplasm and provide a list of RAS protein interactions with possible cancer-related consequences, which could help guide future therapeutic strategies to target RAS proteins.
    [Show full text]
  • Lipid Signaling in T-Cell Development and Function
    Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Lipid Signaling in T-Cell Development and Function Yina H. Huang1 and Karsten Sauer2 1Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 2Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037 Correspondence: [email protected] Second messenger molecules relay, amplify, and diversify cell surface receptor signals. Two important examples are phosphorylated D-myo-inositol derivatives, such as phosphoinosi- tide lipids within cellular membranes, and soluble inositol phosphates. Here, we review how phosphoinositide metabolism generates multiple second messengers with important roles in T-cell development and function. They include soluble inositol(1,4,5)trisphosphate, long known for its Ca2þ-mobilizing function, and phosphatidylinositol(3,4,5)trisphosphate, whose generation by phosphoinositide 3-kinase and turnover by the phosphatases PTEN and SHIP control a key “hub” of TCR signaling. More recent studies unveiled important second messenger functions for diacylglycerol, phosphatidic acid, and soluble inositol(1,3,4,5)tetra- kisphosphate (IP4) in immune cells. Inositol(1,3,4,5)tetrakisphosphate acts as a soluble phosphatidylinositol(3,4,5)trisphosphate analog to control protein membrane recruitment. We propose that phosphoinositide lipids and soluble inositol phosphates (IPs) can act as complementary partners whose interplay could have broadly important roles in cellular signaling. econd messenger molecules relay, amplify, phosphoinositides and soluble IPs. Many of Sand diversify signals perceived by cell surface these regulate distinct and overlapping down- receptors. One important second messenger stream effectors (Irvine and Schell 2001; Alca- family is comprised of the phosphorylated der- zar-Roman and Wente 2008; Resnick and ivatives of the small cyclic poly-alcohol D- Saiardi 2008; Sauer et al.
    [Show full text]
  • Deconstruction of the Ras Switching Cycle Through Saturation Mutagenesis
    Deconstruction of the Ras switching cycle through saturation mutagenesis The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Bandaru, Pradeep et al. “Deconstruction of the Ras Switching Cycle through Saturation Mutagenesis.” eLife 2017, 6 (July 2017): e27810 © Bandaru et al As Published http://dx.doi.org/10.7554/ELIFE.27810 Publisher eLife Sciences Publications, Ltd Version Final published version Citable link http://hdl.handle.net/1721.1/113564 Terms of Use Creative Commons Attribution 4.0 International License Detailed Terms https://creativecommons.org/licenses/by/4.0/ RESEARCH ARTICLE Deconstruction of the Ras switching cycle through saturation mutagenesis Pradeep Bandaru1,2,3, Neel H Shah1,2,3, Moitrayee Bhattacharyya1,2,3, John P Barton4,5,6,7, Yasushi Kondo1,2,3, Joshua C Cofsky1,2,3, Christine L Gee1,2,3, Arup K Chakraborty4,5,6,7,8,9, Tanja Kortemme10, Rama Ranganathan11,12,13*, John Kuriyan1,2,3,14* 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; 2California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States; 3Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; 4Ragon Institute of MGH, MIT and Harvard, Cambridge, United States; 5Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, United States; 6Department of Physics, Massachusetts Institute of Technology, Cambridge, United
    [Show full text]
  • Essential Gene Profiles in Breast, Pancreatic, and Ovarian Cancer Cells
    Published OnlineFirst December 29, 2011; DOI: 10.1158/2159-8290.CD-11-0224 ReseaRch aRticLe Essential Gene Profiles in Breast, Pancreatic, and Ovarian Cancer Cells Richard Marcotte4, Kevin R. Brown1, Fernando Suarez4, Azin Sayad1, Konstantina Karamboulas1, Paul M. Krzyzanowski4, Fabrice Sircoulomb4, Mauricio Medrano3,4, Yaroslav Fedyshyn1, Judice L.Y. Koh1, Dewald van Dyk1, Bohdana Fedyshyn1, Marianna Luhova1, Glauber C. Brito1, Franco J. Vizeacoumar1, Frederick S. Vizeacoumar5, Alessandro Datti5,7, Dahlia Kasimer1, Alla Buzina1, Patricia Mero1, Christine Misquitta1, Josee Normand4, Maliha Haider4, Troy Ketela1, Jeffrey L. Wrana2,5, Robert Rottapel3,4,6, Benjamin G. Neel3,4, and Jason Moffat1,2 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst December 29, 2011; DOI: 10.1158/2159-8290.CD-11-0224 The BATTLE Trial: Personalizing Therapy for Lung Cancer rEsEarCh artiClE a bstRact Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive de- scription of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer pheno- type (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identifi- cation of the genes essential for cancer cell survival and proliferation, providing a “functional ge- nomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines.
    [Show full text]