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Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis*

  • Kazutaka Sumita
    Footnotes
    Affiliations
    Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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  • Hirofumi Yoshino
    Footnotes
    Affiliations
    Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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  • Mika Sasaki
    Footnotes
    Affiliations
    Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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  • Nazanin Majd
    Affiliations
    Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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  • Emily Rose Kahoud
    Affiliations
    Department of Medicine, Harvard Medical School, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
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  • Hidenori Takahashi
    Affiliations
    Department of Medicine, Harvard Medical School, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
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  • Koh Takeuchi
    Affiliations
    Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-3-26 Aomi, Koto, Tokyo 135-0064, Japan
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  • Taruho Kuroda
    Affiliations
    Department of Basic Medical Sciences, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
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  • Susan Lee
    Affiliations
    Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0380
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  • Pascale G. Charest
    Affiliations
    Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0380
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  • Kosuke Takeda
    Affiliations
    Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0380

    Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore, 138667, Republic of Singapore
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  • John M. Asara
    Affiliations
    Department of Medicine, Harvard Medical School, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
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  • Richard A. Firtel
    Affiliations
    Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0380
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  • Dimitrios Anastasiou
    Affiliations
    Division of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
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  • Atsuo T. Sasaki
    Correspondence
    To whom correspondence should be addressed: UC Cancer Inst., Dept. of Internal Medicine, Div. of Hematology Oncology, UC Neuroscience Institute, Dept. of Neurosurgery, Brain Tumor Center, University of Cincinnati, College of Medicine, Cincinnati, OH 45267. Tel.: 513-558-2160; Fax: 513-558-6703
    Affiliations
    Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants 2P01CA120964 (to J. M. A.) and 5P30CA006516 (to J. M. A.) and the University of Cincinnati (to A. T. S.).
    1 These authors contributed equally to this work.
    2 Supported in part by the American Association of Neurological Surgeons.
Open AccessPublished:December 13, 2013DOI:https://doi.org/10.1074/jbc.M113.531178
      Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.

      Introduction

      Ras is a small GTPase that can cycle between a GTP-bound active state and a GDP-bound inactive state. GTP-loaded Ras elicits an array of downstream signaling such as Raf/MEK/ERK and PI3K/AKT pathways to promote cell survival and proliferation (
      • Quinlan M.P.
      • Quatela S.E.
      • Philips M.R.
      • Settleman J.
      Activated Kras, but not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion.
      ,
      • Karnoub A.E.
      • Weinberg R.A.
      Ras oncogenes: split personalities.
      ,
      • Haigis K.M.
      • Kendall K.R.
      • Wang Y.
      • Cheung A.
      • Haigis M.C.
      • Glickman J.N.
      • Niwa-Kawakita M.
      • Sweet-Cordero A.
      • Sebolt-Leopold J.
      • Shannon K.M.
      • Settleman J.
      • Giovannini M.
      • Jacks T.
      Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
      ,
      • Wennerberg K.
      • Rossman K.L.
      • Der C.J.
      The Ras superfamily at a glance.
      ,
      • Vetter I.R.
      • Wittinghofer A.
      The guanine nucleotide-binding switch in three dimensions.
      ).
      Mammalian cells encode four closely related Ras proteins, H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B that are highly homologous in the first 85% of the protein and share a C-terminal CAAX box that targets them for farnesylation. The C-terminal hypervariable region (HVR)
      The abbreviations used are:
      HVR
      hypervariable region
      CHX
      cycloheximide
      7KR-RasG
      seven lysine residues in the membrane-targeting region substituted for arginine residues
      4KR-RasG
      four lysines found to be ubiquitinated are substituted with arginines.
      renders distinctive roles of Ras isoforms. The HVR of K-Ras 4B (hereafter called K-Ras), a dominant K-Ras isoform, contains a poly-basic, lysine-rich, cluster that is required for K-Ras plasma membrane localization. The HVRs of the other Ras isoforms, including K-Ras 4A, another minor K-Ras isoform, provide palmitoylation site(s) for targeting to the plasma membrane (
      • Downward J.
      Targeting RAS signalling pathways in cancer therapy.
      ,
      • Hancock J.F.
      Ras proteins: different signals from different locations.
      ,
      • Malumbres M.
      • Barbacid M.
      RAS oncogenes: the first 30 years.
      ,
      • Prior I.A.
      • Hancock J.F.
      Ras trafficking, localization and compartmentalized signalling.
      ,
      • Mor A.
      • Philips M.R.
      Compartmentalized ras/mapk signaling.
      ). Extensive research within the past two decades has shown that Ras isoforms differ in their ability to activate downstream effectors and promote transformation (
      • Sasaki A.T.
      • Chun C.
      • Takeda K.
      • Firtel R.A.
      Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.
      ,
      • Keller J.W.
      • Haigis K.M.
      • Franklin J.L.
      • Whitehead R.H.
      • Jacks T.
      • Coffey R.J.
      Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS: gelsolin complex.
      ,
      • Voice J.K.
      • Klemke R.L.
      • Le A.
      • Jackson J.H.
      Four human ras homologs differ in their abilities to activate Raf-1, induce transformation, and stimulate cell motility.
      ). In mouse tumor models, only the constitutively active K-Ras mutant, but not H-Ras or N-Ras mutants, promotes colorectal carcinogenesis and hyperproliferation of endoderm progenitor cells (
      • Quinlan M.P.
      • Quatela S.E.
      • Philips M.R.
      • Settleman J.
      Activated Kras, but not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion.
      ,
      • Haigis K.M.
      • Kendall K.R.
      • Wang Y.
      • Cheung A.
      • Haigis M.C.
      • Glickman J.N.
      • Niwa-Kawakita M.
      • Sweet-Cordero A.
      • Sebolt-Leopold J.
      • Shannon K.M.
      • Settleman J.
      • Giovannini M.
      • Jacks T.
      Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
      ,
      • Sasaki A.T.
      • Carracedo A.
      • Locasale J.W.
      • Anastasiou D.
      • Takeuchi K.
      • Kahoud E.R.
      • Haviv S.
      • Asara J.M.
      • Pandolfi P.P.
      • Cantley L.C.
      Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors.
      ,
      • Spoerner M.
      • Herrmann C.
      • Vetter I.R.
      • Kalbitzer H.R.
      • Wittinghofer A.
      Dynamic properties of the Ras switch I region and its importance for binding to effectors.
      ). In human cancers, K-Ras is the most frequently mutated Ras isoform. Furthermore, K-Ras mutations are associated with poor clinical prognosis and resistance to chemotherapy (
      • Downward J.
      Targeting RAS signalling pathways in cancer therapy.
      ,
      • Malumbres M.
      • Barbacid M.
      RAS oncogenes: the first 30 years.
      ,
      • Khosla M.
      • Spiegelman G.B.
      • Weeks G.
      Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.
      ,
      • Thiery R.
      • Robbins S.
      • Khosla M.
      • Spiegelman G.B.
      • Weeks G.
      The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium.
      ,
      • Jaffer Z.M.
      • Khosla M.
      • Spiegelman G.B.
      • Weeks G.
      Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.
      ). Despite numerous attempts, a small molecule inhibitor for K-Ras is yet to be developed for clinical use. Identification of KRas-specific regulatory mechanisms is likely to provide important insights into the development of novel therapeutic applications for K-Ras-mutated tumors.
      To date, many of the physiological roles of Ras have been identified through studies using model organisms. However, not all model organisms have both H-Ras and K-Ras isoforms. Saccharomyces cerevisiae, a unicellular organism, has H-Ras homologue genes Ras1 and Ras2, but it does not encode for a K-Ras orthologue. Caenorhabditis elegans and Drosophila melanogaster have a single K-Ras orthologous gene but not an H-Ras orthologue. Dictyostelium discoideum, a social amoeba that has the ability to alternate between a unicellular and a multicellular form, has both K-Ras and H-Ras orthologues. Therefore, to investigate whether ubiquitination comprises an evolutionarily conserved mechanism for Ras regulation, we utilized the Dictyostelium system.

      Acknowledgments

      We gratefully acknowledge the members of the University of Cincinnati Cancer Institute, the UC Brain Tumor Center and the Izayoi Meeting for stimulating discussions and helpful suggestions, Dr. Melanie Cushion, Dr. Antonis Koromilas, Mary Kemper, Dillon Chen, and Janice Connelly for help in critical reading and preparing this manuscript. We sincerely thank Dr. Lewis Cantley for the critical comments, support, and encouragement. We also thank Drs. Ronald Warnick, Sarah Kozma, Carol Mercer, and George Thomas for generous support and feedback. We thank Xuemei Yang and Min Yuan for help with mass spectrometry experiments.

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