Evidence for Mutation-Specific Selectivity in Non-Small Cell
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Localization of Ralb Signaling at Endomembrane Compartments and Its Modulation by Autophagy Received: 14 January 2019 Manish Kumar Singh1,2, Alexandre P
www.nature.com/scientificreports Corrected: Publisher Correction OPEN Localization of RalB signaling at endomembrane compartments and its modulation by autophagy Received: 14 January 2019 Manish Kumar Singh1,2, Alexandre P. J. Martin1,2, Carine Jofre 3, Giulia Zago1,2, Accepted: 30 May 2019 Jacques Camonis1,2, Mathieu Coppey1,4 & Maria Carla Parrini1,2 Published online: 20 June 2019 The monomeric GTPase RalB controls crucial physiological processes, including autophagy and invasion, but it still remains unclear how this multi-functionality is achieved. Previously, we reported that the RalGEF (Guanine nucleotide Exchange Factor) RGL2 binds and activates RalB to promote invasion. Here we show that RGL2, a major activator of RalB, is also required for autophagy. Using a novel automated image analysis method, Endomapper, we quantifed the endogenous localization of the RGL2 activator and its substrate RalB at diferent endomembrane compartments, in an isogenic normal and Ras-transformed cell model. In both normal and Ras-transformed cells, we observed that RGL2 and RalB substantially localize at early and recycling endosomes, and to lesser extent at autophagosomes, but not at trans-Golgi. Interestingly the use of a FRET-based RalB biosensor indicated that RalB signaling is active at these endomembrane compartments at basal level in rich medium. Furthermore, induction of autophagy by nutrient starvation led to a considerable reduction of early and recycling endosomes, in contrast to the expected increase of autophagosomes, in both normal and Ras-transformed cells. However, autophagy mildly afected relative abundances of both RGL2 and RalB at early and recycling endosomes, and at autophagosomes. Interestingly, RalB activity increased at autophagosomes upon starvation in normal cells. -
The Role of Rala and Ralb in Cancer Samuel C
University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 4-7-2008 The Role of RalA and RalB in Cancer Samuel C. Falsetti University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Falsetti, Samuel C., "The Role of RalA and RalB in Cancer" (2008). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/232 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The Role of RalA and RalB in Cancer By Samuel C. Falsetti A dissertation submitted in partial fulfillment Of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Saïd M. Sebti, Ph.D. Larry P. Solomonson, Ph.D. Gloria C. Ferreira, Ph.D. Srikumar Chellapan, Ph.D. Gary Reuther, Ph.D. Douglas Cress, Ph.D. Date of Approval: April 7th, 2008 Keywords: Ras, RACK1, Geranylgeranyltransferase I inhibitors, ovarian cancer, proteomics © Copyright 2008, Samuel C. Falsetti Dedication This thesis is dedicated to my greatest supporter, my wife. Without her loving advice and patience none of this would be possible. Acknowledgments I would like to extend my sincere gratitude to my wife, Nicole. She is the most inspirational person in my life and I am honored to be with her; in truth, this degree ought to come with two names printed on it. -
Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002) -
RAP2 Mediates Mechanoresponses of the Hippo Pathway Zhipeng Meng1, Yunjiang Qiu2,3, Kimberly C
LETTER https://doi.org/10.1038/s41586-018-0444-0 RAP2 mediates mechanoresponses of the Hippo pathway Zhipeng Meng1, Yunjiang Qiu2,3, Kimberly C. Lin1, Aditya Kumar4, Jesse K. Placone4, Cao Fang1, Kuei-Chun Wang4,5, Shicong Lu1, Margaret Pan1, Audrey W. Hong1, Toshiro Moroishi1,6,7, Min Luo1,8, Steven W. Plouffe1, Yarui Diao2, Zhen Ye2, Hyun Woo Park1,9, Xiaoqiong Wang10, Fa-Xing Yu11, Shu Chien4,5, Cun-Yu Wang12, Bing Ren2,13, Adam J. Engler4 & Kun-Liang Guan1* Mammalian cells are surrounded by neighbouring cells and Extended Data Fig. 1b). Expression of the YAP and TAZ target extracellular matrix (ECM), which provide cells with structural genes CTGF, CYR61, and ANKRD1 was repressed by low stiffness in support and mechanical cues that influence diverse biological wild-type cells, but not in RAP2-KO cells (Fig. 1f). Similar results were processes1. The Hippo pathway effectors YAP (also known as YAP1) observed in human mesenchymal stem cells (Extended Data Fig. 1c–e), and TAZ (also known as WWTR1) are regulated by mechanical in which RAP2 deletion suppressed differentiation into adipocytes cues and mediate cellular responses to ECM stiffness2,3. Here we (Extended Data Fig. 1f, g). In luminal breast cancer MCF7 cells, identified the Ras-related GTPase RAP2 as a key intracellular ECM stiffness modulated localization of YAP and TAZ in a RAP2- signal transducer that relays ECM rigidity signals to control dependent manner, whereas the basal type MDA-MB-468 cells showed mechanosensitive cellular activities through YAP and TAZ. constitutively cytoplasmic localization of YAP and TAZ regardless RAP2 is activated by low ECM stiffness, and deletion of RAP2 of stiffness (Extended Data Fig. -
SHOC2–MRAS–PP1 Complex Positively Regulates RAF Activity and Contributes to Noonan Syndrome Pathogenesis
SHOC2–MRAS–PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis Lucy C. Younga,1, Nicole Hartiga,2, Isabel Boned del Ríoa, Sibel Saria, Benjamin Ringham-Terrya, Joshua R. Wainwrighta, Greg G. Jonesa, Frank McCormickb,3, and Pablo Rodriguez-Vicianaa,3 aUniversity College London Cancer Institute, University College London, London WC1E 6DD, United Kingdom; and bHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158 Contributed by Frank McCormick, September 18, 2018 (sent for review November 22, 2017; reviewed by Deborah K. Morrison and Marc Therrien) Dephosphorylation of the inhibitory “S259” site on RAF kinases CRAF/RAF1 mutations are also frequently found in NS and (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. cluster around the S259 14-3-3 binding site, enhancing CRAF ac- The MRAS GTPase, a close relative of RAS oncoproteins, interacts tivity through disruption of 14-3-3 binding (8) and highlighting the with SHOC2 and protein phosphatase 1 (PP1) to form a heterotri- key role of this regulatory step in RAF–ERK pathway activation. meric holoenzyme that dephosphorylates this S259 RAF site. MRAS is a very close relative of the classical RAS oncoproteins MRAS and SHOC2 function as PP1 regulatory subunits providing (H-, N-, and KRAS, hereafter referred to collectively as “RAS”) the complex with striking specificity against RAF. MRAS also func- and shares most regulatory and effector interactions as well as tions as a targeting subunit as membrane localization is required transforming ability (9–11). However, MRAS also has specific for efficient RAF dephosphorylation and ERK pathway regulation functions of its own, and uniquely among RAS family GTPases, it in cells. -
Phosphorylation of Ralb Is Important for Bladder Cancer Cell Growth and Metastasis
Published OnlineFirst October 12, 2010; DOI: 10.1158/0008-5472.CAN-10-0952 Published OnlineFirst on October 12, 2010 as 10.1158/0008-5472.CAN-10-0952 Therapeutics, Targets, and Chemical Biology Cancer Research Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis Hong Wang1, Charles Owens1, Nidhi Chandra1, Mark R. Conaway2, David L. Brautigan3,4, and Dan Theodorescu5 Abstract RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paralogue- specific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphosite- specific antibodies, here we show phosphorylation of RalB by protein kinase C (PKC) and RalA by protein kinase A. We used mass spectrometry and site-directed mutagenesis to identify S198 as the primary PKC phosphorylation site in RalB. Phorbol ester [phorbol 12-myristate 13-acetate (PMA)] treatment of human blad- der carcinoma cells induced S198 phosphorylation of stably expressed FLAG-RalB as well as endogenous RalB. PMA treatment caused RalB translocation from the plasma membrane to perinuclear regions in a S198 phos- phorylation–dependent manner. Using RNA interference depletion of RalB followed by rescue with wild-type RalB or RalB(S198A) as well as overexpression of wild-type RalB or RalB(S198A) with and without PMA stimulation, we show that phosphorylation of RalB at S198 is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis of T24 or UMUC3 human bladder cancer cells. In addition, UMUC3 cells transfected with a constitutively active RalB(G23V) exhibited enhanced subcutaneous tumor growth, whereas those transfected with phospho-deficient RalB(G23V-S198A) were indistinguishable from control cells. -
Identification of the Rassf3 Gene As a Potential Tumor Suppressor
Clemson University TigerPrints All Dissertations Dissertations 12-2006 Identification of the Rassf3 Gene as a Potential Tumor Suppressor Responsible for the Resistance to Mammary Tumor Development in MMTV/ neu Transgenic Mice Isabelle Jacquemart Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Microbiology Commons Recommended Citation Jacquemart, Isabelle, "Identification of the Rassf3 Gene as a Potential Tumor Suppressor Responsible for the Resistance to Mammary Tumor Development in MMTV/neu Transgenic Mice" (2006). All Dissertations. 26. https://tigerprints.clemson.edu/all_dissertations/26 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. IDENTIFICATION OF THE Rassf3 GENE AS A POTENTIAL TUMOR SUPPRESSOR RESPONSIBLE FOR THE RESISTANCE TO MAMMARY TUMOR DEVELOPMENT IN MMTV/neu TRANSGENIC MICE A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Microbiology by Isabelle C. Jacquemart December 2006 Accepted by: Dr. Wen Y. Chen, Committee Chair Dr. Charles D. Rice Dr. Lyndon L. Larcom Dr. Lesly Temesvari i ABSTRACT The MMTV/neu transgenic mouse line is a well-documented animal model for studying HER2/neu-related breast cancer. It has been reported that a small percentage, approximately 20%, of the virgin female MMTV/neu mice seems resistant to the development of mammary gland adenoma, despite the overexpression of the neu oncogene. To identify the factors that are responsible for the tumor resistance in these MMTV/neu female transgenic mice, comparative genetic profiling was used to screen the alterations in gene expression in the mammary gland. -
The Small G-Protein Rala Promotes Progression and Metastasis of Triple- Negative Breast Cancer Katie A
Thies et al. Breast Cancer Research (2021) 23:65 https://doi.org/10.1186/s13058-021-01438-3 RESEARCH ARTICLE Open Access The small G-protein RalA promotes progression and metastasis of triple- negative breast cancer Katie A. Thies1,2, Matthew W. Cole1,2, Rachel E. Schafer1,2, Jonathan M. Spehar1,2, Dillon S. Richardson1,2, Sarah A. Steck1,2, Manjusri Das1,2, Arthur W. Lian1,2, Alo Ray1,2, Reena Shakya1,3, Sue E. Knoblaugh4, Cynthia D. Timmers5,6, Michael C. Ostrowski5,7, Arnab Chakravarti1,2, Gina M. Sizemore1,2 and Steven T. Sizemore1,2* Abstract Background: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. -
Genetic Deletion of RALA and RALB Small Gtpases Reveals Redundant Functions in Development and Tumorigenesis
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 22, 2063–2068, November 6, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.09.013 Report Genetic Deletion of RALA and RALB Small GTPases Reveals Redundant Functions in Development and Tumorigenesis Pascal Peschard,1 Afshan McCarthy,1 (Figures 1A and 1B) and loss of RALB proteins in tissues of Vale´rie Leblanc-Dominguez,1 Maggie Yeo,1 adult mice (Figure 1C). The expression of RALB proteins was Sabrina Guichard,1 Gordon Stamp,2 unchanged in Rala null cells and vice versa. and Christopher J. Marshall1,* To investigate the role of RALA in embryonic development, 1Oncogene Team, Division of Cancer Biology, Institute of we examined embryos from Rala+/2 intercrosses. Between Cancer Research, London SW3 6JB, UK embryonic day 10.5 (E10.5) and E19.5, we observed that 10 2Department of Histopathology, Royal Marsden Hospital, out of 33 of the Rala null embryos displayed exencephaly London SW3 6JJ, UK (Figures 1E and 1F), a condition caused by a failure of closure of the neural tube along the hindbrain region [15]. We also observed exencephaly in Rala null embryos generated from Summary a gene-trap embryonic stem (ES) clone (Figures 1G and S1E– S1I). To examine whether RALA and RALB have overlapping RAL small GTPases, encoded by the Rala and Ralb genes, functions in neural tube closure, we generated compound are members of the RAS superfamily of small GTPases and mutants: 14 of 14 Rala2/2;Ralb+/2 embryos displayed a severe can act as downstream effectors of RAS [1]. -
Targeting the Ras-Ral Effector Pathway for Cancer Treatment
TARGETING THE RAS-RAL EFFECTOR PATHWAY FOR CANCER TREATMENT Leanna R. Gentry A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2015 Approved by: Adrienne Cox Channing Der Lee Graves Klaus Hahn Gary Johnson ©2015 Leanna R. Gentry ALL RIGHTS RESERVED ii ABSTRACT LEANNA R. GENTRY: Targeting the Ras-Ral effector pathway for cancer treatment (Under the direction of Channing J. Der) The RAS oncogene is the most frequently mutated gene in human cancers, and this activated Ras oncoprotein has been shown to be required for both cancer initiation and maintenance. Great strides have been made in understanding Ras signaling in cancer since the discovery of its involvement in human cancers in 1982, with numerous Ras effector pathways and modes of Ras regulation having been identified as contributing to Ras-driven oncogenesis. However, there has been limited success in developing strategies for therapeutically targeting Ras-driven oncogenesis. One effort that has gained popularity in recent years is the inhibition of Ras effector signaling. The Ral (Ras-like) small GTPases, discovered shortly after Ras in an attempt to identify RAS-related genes, are activated downstream of Ras by Ral guanine nucleotide exchange factors (RalGEFs). The Ral family members have since emerged as critical regulators of key cellular processes and, importantly, have been characterized as playing a role in tumorigenesis and invasion of multiple cancer types. Interestingly, divergent roles for RalA and RalB are often observed in within a cancer. -
The RAS–Effector Interaction As a Drug Target Adam B
Published OnlineFirst January 6, 2017; DOI: 10.1158/0008-5472.CAN-16-0938 Cancer Review Research The RAS–Effector Interaction as a Drug Target Adam B. Keeton1,2, E. Alan Salter3, and Gary A. Piazza1,2 Abstract About a third of all human cancers harbor mutations in one direct inhibitors of RAS proteins. Here, we review this evidence of the K-, N-, or HRAS genes that encode an abnormal RAS with a focus on compounds capable of inhibiting the interac- protein locked in a constitutively activated state to drive malig- tion of RAS proteins with their effectors that transduce the nant transformation and tumor growth. Despite more than signals of RAS and that drive and sustain malignant transfor- three decades of intensive research aimed at the discovery of mation and tumor growth. These reports of direct-acting RAS RAS-directed therapeutics, there are no FDA-approved drugs inhibitors provide valuable insight for further discovery and that are broadly effective against RAS-driven cancers. Although development of clinical candidates for RAS-driven cancers RAS proteins are often said to be "undruggable," there is involving mutations in RAS genes or otherwise activated RAS mounting evidence suggesting it may be feasible to develop proteins. Cancer Res; 77(2); 221–6. Ó2017 AACR. Introduction Activating mutations at codons 12, 13, or 61 of KRAS occur de novo in approximately one third of all human cancers and are Cancer is a leading cause of death in the developed world with especially prevalent in pancreatic, colorectal, and lung tumors. more than one million people diagnosed and more than 500,000 These mutations affect the P-loop and switch-2 regions of the deaths per year in the United States alone. -
Exocyst Inactivation in Urothelial Cells Disrupts Autophagy and Activates Non-Canonical
bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451173; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Exocyst Inactivation in Urothelial Cells Disrupts Autophagy and Activates non-canonical NF-κB Michael A. Ortega1,2, Ross K. Villiger2, Malia Harrison-Chau2, Suzanna Lieu2, Kadee-Kalia Tamashiro2, Amanda J. Lee2,3, Brent A. Fujimoto2, Geetika Y. Patwardhan2, Joshua Kepler2 & Ben Fogelgren*2 1 Center for Biomedical Research at The Queen’s Medical Center, Honolulu, Hawaii 96813; 2 Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813; 3 Math and Sciences Department, Kapiolani Community College, Honolulu, Hawaii 96816. *Corresponding Author: Ben Fogelgren Email address: [email protected] Keywords: autophagy / exocyst / Fn14 / NF-κB / urothelium 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451173; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Ureter obstruction is a highly prevalent event during embryonic development and is a major cause of pediatric kidney disease. We have reported that ureteric bud-specific ablation of the exocyst Exoc5 subunit in late-murine gestation results in failure of urothelial stratification, cell death, and complete ureter obstruction. However, the mechanistic connection between disrupted exocyst activity, urothelial cell death, and subsequent ureter obstruction was unclear. Here, we report that inhibited urothelial stratification does not drive cell death during ureter development.