DOCSLIB.ORG

Regulation of Ras and Rho Gtpases by Post-Translational Modification: Cysteine Oxidation and Ubiquitination

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Ras and Rho Gtpases by Post-Translational Modification: Cysteine Oxidation and Ubiquitination REGULATION OF RAS AND RHO GTPASES BY POST-TRANSLATIONAL MODIFICATION: CYSTEINE OXIDATION AND UBIQUITINATION Guy Aaron Hobbs A dissertation submitted to the faculty of 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 Biochemistry and Biophysics. Chapel Hill 2013 Approved by: Sharon Campbell Henrik Dohlman Brian Kuhlman Keith Burridge Andrew Lee © 2013 Guy Aaron Hobbs ALL RIGHTS RESERVED ii ABSTRACT Guy Aaron Hobbs: Regulation of Ras and Rho GTPases by Post-Translational Modification: Cysteine Oxidation and Ubiquitination (Under the direction of Sharon L. Campbell) Ras superfamily GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras superfamily GTPases is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. In addition, evidence for the redox regulation of Ras superfamily GTPases is growing, and current data supports regulation by the generation of a thiyl radical. Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. In Ras GTPases, the redox-sensitive cysteine is in the NKCD motif, which is a motif that is critical for nucleotide binding. However, we show that oxidation that occurs absent of the generation of a thiyl radical does not result in Ras activation. In the Rho subfamily of GTPases, there is increasing in vitro and cell-based data supporting the oxidant-mediated regulation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop motif (GXXXXG[S/T]C). While this motif is distinct from Ras, our data suggest that Rho GTPases are regulated by free radical-generation as well as oxidation of the thiol. Specifically for RhoA and other Rho GTPases that have two cysteine residues within the phosphoryl-binding loop motif (GXXCXG[S/T]C), oxidation can result in internal disulfide bond formation that can inactivate these GTPases. In addition, we characterized relevant cellular oxidation states of RhoA and show that oxidation can result in the activation and deactivation of RhoA. iii Thus, from data presented herein, it is evident that Ras and Rho family GTPases are uniquely regulated by free radical and non-radical oxidants, and the modulation of activity of the GTPase due to oxidation is dependent on the position(s) of the redox-sensitive thiol. While Ras GTPases are only activated by free radicals because the redox-sensitive thiol does not make interactions with the bound nucleotide, in Rho GTPases, the redox-sensitive thiol makes direct interactions with the bound nucleotide, which results in activation upon modification of the redox-sensitive thiol in RhoA and related GTPases. iv ACKNOWLEDGEMENTS Foremost, I would like to acknowledge the individuals that have encouraged me always question the world around me. I would specifically like to acknowledge Dr. Gloria Lee for giving me a chance to work in her lab as an undergraduate. At the bench, Dr. Kiran Bhaskar and Dr. Daniel Ferraro were my first mentors in research while an undergraduate student at the University of Iowa. In addition, I would like to acknowledge Dr. Ramaswamy Subramanian and Dr. Bryce Plapp for their guidance and mentorship. I do not think I would be in the place I am today without the care and patience of these individuals. I would also like to acknowledge my mentor, Dr. Sharon Campbell, for always being there to listen to my wild ideas and provide feedback on new data. Her willingness to discuss anything will be greatly missed. While others got me excited to perform research, Sharon taught me how to think like a scientist. To specifically acknowledge every person that helped along the way would result in an acknowledgements section as long as the dissertation itself. Thus, I say thank you to current and previous lab members, classmates, and friends for asking questions, challenging my thoughts, and providing support when needed. Minh Huynh, who was an undergraduate student under my direction, served with me for two years and deserves a special acknowledgement. He ran every project during the last two years of my thesis by preparing proteins, buffers, and reagents, often with minimal direction, which allowed me the time to perform the most critical experiments as well as analyze and present the data. Without his help, the amount that I was able to accomplish would be significantly less. Lastly, I want to thank my parents and family for encouraging and fostering a desire to read, learn, and take my education seriously. Their unwavering support and v encouragement to achieve good grades, instill a solid work ethic, and do what I loved has led me to be who I am today. There are no words to describe the level of gratitude I have for my parents for teaching me right from wrong as well as the importance of never quitting and doing your best. I also wish the thank Heather. Although she joined me in my last two years of my journey at UNC, they were the most productive two years as well as the most exciting years. She taught me what school could never teach a person, led me out of the lab on some amazing adventures, and let me back into the lab to complete my work. While she has captured my heart, I will never be able to thank her enough for her help and support through some of the most demanding years in graduate school. Lastly, I must thank Josh Gough for providing me with one of my favorite sayings, “It sucks to suck.” It truly does suck to suck, and this saying has repeatedly served as motivation and as words of encouragement. Thank you. vi TABLE OF CONTENTS LIST OF FIGURES............................................................................................................... .... x LIST OF TABLES ...................................................................................................................xii LIST OF ABBREVIATIONS AND SYMBOLS ..................................................................... xiii Chapter 1: Introduction and Background ............................................................................... 1 Introduction ................................................................................................................. 1 Generation of ROS and RNS in cells and antioxidant defense ................................. 4 Reactive oxygen and nitrogen species as second messengers .................................. 9 Free radicals modify and activate Ras through a reactive cysteine in the NKCD motif ............................................................................................. 11 Redox regulation of Rho GTPases by 1e- and 2e- mechanisms through a redox-active cysteine in the phosphoryl-binding loop ................................................................................................................ 14 Chapter 2: Glutathiolated Ras: Characterization and Implications for Ras Activation ................................................................................................................... 17 Introduction ............................................................................................................... 17 Materials and Methods ............................................................................................ 20 Results ...................................................................................................................... 24 Discussion ................................................................................................................. 32 Conclusions ............................................................................................................... 37 Chapter 3: Characterization and Nitrosation of Oncogenic K-RasG12C ............................... 40 Introduction .............................................................................................................. 40 Methods .................................................................................................................... 42 vii Results ...................................................................................................................... 44 Discussion ................................................................................................................. 52 Chapter 4: Oxidation of RhoA at Cys20 regulates nucleotide binding ................................ 56 Introduction .............................................................................................................. 56 Methods .................................................................................................................... 60 Results ...................................................................................................................... 64 Discussion ................................................................................................................. 82 Chapter 5: Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function ......................................................................... 88 Introduction .............................................................................................................. 88 Commentary ............................................................................................................. 88 Chapter 6: Biophysical
Load more

Recommended publications
  • The Rac Gtpase in Cancer: from Old Concepts to New Paradigms Marcelo G
    Published OnlineFirst August 14, 2017; DOI: 10.1158/0008-5472.CAN-17-1456 Cancer Review Research The Rac GTPase in Cancer: From Old Concepts to New Paradigms Marcelo G. Kazanietz1 and Maria J. Caloca2 Abstract Rho family GTPases are critical regulators of cellular func- mislocalization of Rac signaling components. The unexpected tions that play important roles in cancer progression. Aberrant pro-oncogenic functions of Rac GTPase-activating proteins also activity of Rho small G-proteins, particularly Rac1 and their challenged the dogma that these negative Rac regulators solely regulators, is a hallmark of cancer and contributes to the act as tumor suppressors. The potential contribution of Rac tumorigenic and metastatic phenotypes of cancer cells. This hyperactivation to resistance to anticancer agents, including review examines the multiple mechanisms leading to Rac1 targeted therapies, as well as to the suppression of antitumor hyperactivation, particularly focusing on emerging paradigms immune response, highlights the critical need to develop ther- that involve gain-of-function mutations in Rac and guanine apeutic strategies to target the Rac pathway in a clinical setting. nucleotide exchange factors, defects in Rac1 degradation, and Cancer Res; 77(20); 5445–51. Ó2017 AACR. Introduction directed toward targeting Rho-regulated pathways for battling cancer. Exactly 25 years ago, two seminal papers by Alan Hall and Nearly all Rho GTPases act as molecular switches that cycle colleagues illuminated us with one of the most influential dis- between GDP-bound (inactive) and GTP-bound (active) forms. coveries in cancer signaling: the association of Ras-related small Activation is promoted by guanine nucleotide exchange factors GTPases of the Rho family with actin cytoskeleton reorganization (GEF) responsible for GDP dissociation, a process that normally (1, 2).
    [Show full text]
  • A GTP-State Specific Cyclic Peptide Inhibitor of the Gtpase Gαs
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.054080; this version posted April 27, 2020. 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. A GTP-state specific cyclic peptide inhibitor of the GTPase Gαs Shizhong A. Dai1,2†, Qi Hu1,2†, Rong Gao3†, Andre Lazar1,4†, Ziyang Zhang1,2, Mark von Zastrow1,4, Hiroaki Suga3*, Kevan M. Shokat1,2* 5 1Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, 94158, USA 2Howard Hughes Medical Institute 3Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 10 4Department of Psychiatry, University of California, San Francisco, San Francisco, CA, 94158, USA *Correspondence to: [email protected], [email protected] †These authors contributed equally. 15 20 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.054080; this version posted April 27, 2020. 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. Abstract: The G protein-coupled receptor (GPCR) cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable receptors and enzymes with the exception of the stimulatory G protein α subunit, Gαs.
    [Show full text]
  • Involvement of the Rho/Rac Family Member Rhog in Caveolar Endocytosis
    Oncogene (2006) 25, 2961–2973 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Involvement of the Rho/Rac family member RhoG in caveolar endocytosis RM Prieto-Sa´ nchez1, IM Berenjeno1 and XR Bustelo Centro de Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer (IBMCC), and Red Tema´tica Cooperativa de Centros de Ca´ncer, CSIC-University of Salamanca, Campus Unamuno, Salamanca, Spain We show here that the GTPase RhoG is involved in transduction, cell polarity, and intercellular communi- caveolar trafficking. Wild-type RhoG moves sequentially cations (Gruenberg, 2001; Conner and Schmid, 2003; Di to the plasma membrane, intracellular vesicles, and the Guglielmo et al., 2003; Felberbaum-Corti et al., 2003; Golgi apparatus along markers of this endocytic pathway. Gonzalez-Gaitan, 2003). The best-known endocytic Such translocation is associated with changes in RhoG pathway is that composed of clathrin-coated vesicles GDP/GTP levels and is highly dependent on lipid raft (Zerial and McBride, 2001; Conner and Schmid, 2003). integrity and on the function of the GTPase dynamin2. In In this case, we have information regarding the addition, the constitutively active RhoGQ61L mutant is internalization, sorting, recycling, fusion, and delivery preferentially located in endocytic vesicles that can be events that participate in this type of endocytosis (Zerial decorated with markers of the caveola-derived endocytic and McBride, 2001; Conner and Schmid, 2003). This pathway. RhoGQ61L, but not the analogous Rac1 mutant pathway has also been molecularly dissected, resulting protein, affects caveola internalization and the subsequent in the characterization of a large number of regulatory delivery of endocytic vesicles to the Golgi apparatus.
    [Show full text]
  • Clinical Utility of Recently Identified Diagnostic, Prognostic, And
    Modern Pathology (2017) 30, 1338–1366 1338 © 2017 USCAP, Inc All rights reserved 0893-3952/17 $32.00 Clinical utility of recently identified diagnostic, prognostic, and predictive molecular biomarkers in mature B-cell neoplasms Arantza Onaindia1, L Jeffrey Medeiros2 and Keyur P Patel2 1Instituto de Investigacion Marques de Valdecilla (IDIVAL)/Hospital Universitario Marques de Valdecilla, Santander, Spain and 2Department of Hematopathology, MD Anderson Cancer Center, Houston, TX, USA Genomic profiling studies have provided new insights into the pathogenesis of mature B-cell neoplasms and have identified markers with prognostic impact. Recurrent mutations in tumor-suppressor genes (TP53, BIRC3, ATM), and common signaling pathways, such as the B-cell receptor (CD79A, CD79B, CARD11, TCF3, ID3), Toll- like receptor (MYD88), NOTCH (NOTCH1/2), nuclear factor-κB, and mitogen activated kinase signaling, have been identified in B-cell neoplasms. Chronic lymphocytic leukemia/small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, Waldenström macroglobulinemia, hairy cell leukemia, and marginal zone lymphomas of splenic, nodal, and extranodal types represent examples of B-cell neoplasms in which novel molecular biomarkers have been discovered in recent years. In addition, ongoing retrospective correlative and prospective outcome studies have resulted in an enhanced understanding of the clinical utility of novel biomarkers. This progress is reflected in the 2016 update of the World Health Organization classification of lymphoid neoplasms, which lists as many as 41 mature B-cell neoplasms (including provisional categories). Consequently, molecular genetic studies are increasingly being applied for the clinical workup of many of these neoplasms. In this review, we focus on the diagnostic, prognostic, and/or therapeutic utility of molecular biomarkers in mature B-cell neoplasms.
    [Show full text]
  • Tumor Markers
    Tumor Markers Alan H.B. Wu, Ph.D. Professor, Laboratory Medicine, UCSF Section Chief, Clinical Chemistry, Toxicology, Pharmacogenomics Laboratory, SFGH Learning objectives • Know the ideal characteristics of a tumor marker • Understand the role of tumor markers for diagnosis and management of patients with cancer. • Know the emerging technologies for tumor markers • Understand the role of tumor markers for therapeutic selection How do we diagnose cancer today? Physical Examination Blood tests CT scans Biopsy Human Prostate Cancer Normal Blood Smear Chronic Myeloid Leukemia Death rates for cancer vs. heart disease New cancer cases per year Cancer Site or Type New Cases Prostate 218,000 Lung 222,500 Breast 207,500 Colorectal 149,000 Urinary system 131,500 Skin 68,770 Pancreas 43,100 Ovarian 22,000 Myeloma 20,200 Thyroid 44,700 Germ Cell 9,000 Types of Tumor Markers • Hormones (hCG; calcitonin; gastrin; prolactin;) • Enzymes (acid phosphatase; alkaline phosphatase; PSA) • Cancer antigen proteins & glycoproteins (CA125; CA 15.3; CA19.9) • Metabolites (norepinephrine, epinephrine) • Normal proteins (thyroglobulin) • Oncofetal antigens (CEA, AFP) • Receptors (ER, PR, EGFR) • Genetic changes (mutations/translocations, etc.) Characteristics of an ideal tumor marker • Specificity for a single type of cancer • High sensitivity and specificity for cancerous growth • Correlation of marker level with tumor size • Homogeneous (i.e., minimal post-translational modifications) • Short half-life in circulation Roles for tumor markers • Determine risk (PSA)
    [Show full text]
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • A Rhog-Mediated Signaling Pathway That Modulates Invadopodia Dynamics in Breast Cancer Cells Silvia M
    © 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 RESEARCH ARTICLE A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells Silvia M. Goicoechea, Ashtyn Zinn, Sahezeel S. Awadia, Kyle Snyder and Rafael Garcia-Mata* ABSTRACT micropinocytosis, bacterial uptake, phagocytosis and leukocyte One of the hallmarks of cancer is the ability of tumor cells to invade trans-endothelial migration (deBakker et al., 2004; Ellerbroek et al., surrounding tissues and metastasize. During metastasis, cancer cells 2004; Jackson et al., 2015; Katoh et al., 2006, 2000; van Buul et al., degrade the extracellular matrix, which acts as a physical barrier, by 2007). Recent studies have revealed that RhoG plays a role in tumor developing specialized actin-rich membrane protrusion structures cell invasion and may contribute to the formation of invadopodia called invadopodia. The formation of invadopodia is regulated by Rho (Hiramoto-Yamaki et al., 2010; Kwiatkowska et al., 2012). GTPases, a family of proteins that regulates the actin cytoskeleton. Invadopodia are actin-rich adhesive structures that form in the Here, we describe a novel role for RhoG in the regulation of ventral surface of cancer cells and allow them to degrade the invadopodia disassembly in human breast cancer cells. Our results extracellular matrix (ECM) (Gimona et al., 2008). Formation of show that RhoG and Rac1 have independent and opposite roles invadopodia involves a series of steps that include the disassembly in the regulation of invadopodia dynamics. We also show that SGEF of focal adhesions and stress fibers, and the relocalization of several (also known as ARHGEF26) is the exchange factor responsible of their components into the newly formed invadopodia (Hoshino for the activation of RhoG during invadopodia disassembly.
    [Show full text]
  • Screening for Tumor Suppressors: Loss of Ephrin PNAS PLUS Receptor A2 Cooperates with Oncogenic Kras in Promoting Lung Adenocarcinoma
    Screening for tumor suppressors: Loss of ephrin PNAS PLUS receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma Narayana Yeddulaa, Yifeng Xiaa, Eugene Kea, Joep Beumera,b, and Inder M. Vermaa,1 aLaboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037; and bHubrecht Institute, Utrecht, The Netherlands Contributed by Inder M. Verma, October 12, 2015 (sent for review July 28, 2015; reviewed by Anton Berns, Tyler Jacks, and Frank McCormick) Lung adenocarcinoma, a major form of non-small cell lung cancer, injections in embryonic skin cells identified several potential tu- is the leading cause of cancer deaths. The Cancer Genome Atlas morigenic factors (14–16). None of the reported studies have analysis of lung adenocarcinoma has identified a large number of performed direct shRNA-mediated high-throughput approaches previously unknown copy number alterations and mutations, re- in adult mice recapitulating the mode of tumorigenesis in humans. quiring experimental validation before use in therapeutics. Here, we Activating mutations at positions 12, 13, and 61 amino acids in describe an shRNA-mediated high-throughput approach to test a set Kirsten rat sarcoma viral oncogene homolog (KRas) contributes of genes for their ability to function as tumor suppressors in the to tumorigenesis in 32% of lung adenocarcinoma patients (2) by background of mutant KRas and WT Tp53. We identified several activating downstream signaling cascades. Mice with the KRasG12D candidate genes from tumors originated from lentiviral delivery of allele develop benign adenomatous lesions with long latency to shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp- develop adenocarcinoma (17, 18).
    [Show full text]
  • High Throughput Strategies Aimed at Closing the GAP in Our Knowledge of Rho Gtpase Signaling
    cells Review High Throughput strategies Aimed at Closing the GAP in Our Knowledge of Rho GTPase Signaling Manel Dahmene 1, Laura Quirion 2 and Mélanie Laurin 1,3,* 1 Oncology Division, CHU de Québec–Université Laval Research Center, Québec, QC G1V 4G2, Canada; [email protected] 2 Montréal Clinical Research Institute (IRCM), Montréal, QC H2W 1R7, Canada; [email protected] 3 Université Laval Cancer Research Center, Québec, QC G1R 3S3, Canada * Correspondence: [email protected] Received: 21 May 2020; Accepted: 7 June 2020; Published: 9 June 2020 Abstract: Since their discovery, Rho GTPases have emerged as key regulators of cytoskeletal dynamics. In humans, there are 20 Rho GTPases and more than 150 regulators that belong to the RhoGEF, RhoGAP, and RhoGDI families. Throughout development, Rho GTPases choregraph a plethora of cellular processes essential for cellular migration, cell–cell junctions, and cell polarity assembly. Rho GTPases are also significant mediators of cancer cell invasion. Nevertheless, to date only a few molecules from these intricate signaling networks have been studied in depth, which has prevented appreciation for the full scope of Rho GTPases’ biological functions. Given the large complexity involved, system level studies are required to fully grasp the extent of their biological roles and regulation. Recently, several groups have tackled this challenge by using proteomic approaches to map the full repertoire of Rho GTPases and Rho regulators protein interactions. These studies have provided in-depth understanding of Rho regulators specificity and have contributed to expand Rho GTPases’ effector portfolio. Additionally, new roles for understudied family members were unraveled using high throughput screening strategies using cell culture models and mouse embryos.
    [Show full text]
  • The Effects of Oncogenic G12d Mutation on K-Ras Structure, Conformation and Dynamics
    bioRxiv preprint doi: https://doi.org/10.1101/178483; this version posted August 19, 2017. 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. THE EFFECTS OF ONCOGENIC G12D MUTATION ON K-RAS STRUCTURE, CONFORMATION AND DYNAMICS Sezen Vatansever1, 2, 3, Zeynep H. Gümüş2, 3*, Burak Erman1* 1Department of Chemical and Biological Engineering, College of Engineering, Koç University, Rumelifeneri Yolu, 34450, Sarıyer, Istanbul, Turkey 2Department of Genetics and Genomics, 3Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029 *Correspondence: [email protected] (B.E.) and [email protected] (Z.H.G.) ABSTRACT K-Ras is the most frequently mutated protein in human tumors. Activating K-Ras mutations drive cancer initiation, progression and drug resistance, directly leading to nearly a million deaths per year. To understand the mechanisms by which mutations alter K-Ras function, we need to understand their effects on protein dynamics. However, despite decades of research, how oncogenic mutations in K-Ras alter its conformation and dynamics remain to be understood. Here, we present how the most recurrent K-Ras oncogenic mutation, G12D, leads to structural, conformational and dynamical changes that lead to constitutively active K- Ras. We have developed a new integrated MD simulation data analysis approach to quantify such changes in a protein and applied it to K-Ras. Our results show that G12D mutation induces strong negative correlations between the fluctuations of SII and those of the P-loop, Switch I (SI) and α3 regions in K-RasG12D.
    [Show full text]
  • Critical Activities of Rac1 and Cdc42hs in Skeletal Myogenesis
    Critical Activities of Rac1 and Cdc42Hs in Skeletal Myogenesis: Antagonistic Effects of JNK and p38 Pathways Mayya Meriane, Pierre Roux, Michael Primig, Philippe Fort, Cécile Gauthier-Rouvière To cite this version: Mayya Meriane, Pierre Roux, Michael Primig, Philippe Fort, Cécile Gauthier-Rouvière. Critical Activities of Rac1 and Cdc42Hs in Skeletal Myogenesis: Antagonistic Effects of JNK and p38 Path- ways. Molecular Biology of the Cell, American Society for Cell Biology, 2000, 11 (8), pp.2513-2528. 10.1091/mbc.11.8.2513. hal-02267479 HAL Id: hal-02267479 https://hal.archives-ouvertes.fr/hal-02267479 Submitted on 26 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - ShareAlike| 4.0 International License Molecular Biology of the Cell Vol. 11, 2513–2528, August 2000 Critical Activities of Rac1 and Cdc42Hs in Skeletal Myogenesis: Antagonistic Effects of JNK and p38 Pathways Mayya Meriane,* Pierre Roux,* Michael Primig,† Philippe Fort,* and Ce´cile Gauthier-Rouvie`re*‡ *Centre de Recherche de Biochimie Macromole´culaire, Centre National de la Recherche Scientifique Unite´ Propre de Recherche 1086, 34293 Montpellier Cedex, France; and †Institut de Ge´ne´tique Humaine, Centre National de la Recherche Scientifique Unite´ Propre de Recherche 1142, 34396 Montpellier Cedex 5, France Submitted January 5, 2000; Revised April 7, 2000; Accepted May 19, 2000 Monitoring Editor: Paul T.
    [Show full text]
  • Agonist-Induced Formation of Unproductive Receptor-G12 Complexes Najeah Okashaha, Shane C
    Agonist-induced formation of unproductive receptor-G12 complexes Najeah Okashaha, Shane C. Wrightb, Kouki Kawakamic, Signe Mathiasend,e,f, Joris Zhoub, Sumin Lua, Jonathan A. Javitchd,e,f, Asuka Inouec, Michel Bouvierb, and Nevin A. Lamberta,1 aDepartment of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912; bInstitute for Research in Immunology and Cancer, Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3T 1J4, Canada; cGraduate School of Pharmaceutical Sciences, Tohoku University, 980-8578 Sendai, Japan; dDepartment of Psychiatry, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032; eDepartment of Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032; and fDivision of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032 Edited by Brian K. Kobilka, Stanford University School of Medicine, Stanford, CA, and approved July 24, 2020 (received for review February 28, 2020) G proteins are activated when they associate with G protein- association does not always promote G protein activation and coupled receptors (GPCRs), often in response to agonist-mediated may in some circumstances inhibit downstream signaling. receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein acti- Results vation and, conversely, that activated GPCRs do not interact with V2R Interacts with G12 Heterotrimers. Conventional GPCR-G pro- G proteins that they do not activate. Here we show that GPCRs can tein coupling is understood as an allosteric interaction where an form agonist-dependent complexes with G proteins that they do agonist-bound active receptor mediates GDP release by stabi- not activate.
    [Show full text]