Regulation of K-Ras by a Farnesyl-electrostatic Switch

Summary

Principal Investigator: MARK REID PHILIPS
Abstract: DESCRIPTION (provided by applicant): Of the three ras genes, kras is most frequently mutated in human cancer. Ras proteins are highly homologous but differ extensively in their C-terminal hypervariable regions that direct post-translational modifications (e.g. farnesylation) and membrane targeting. We discovered a post-translational modification unique to K-Ras: protein kinase C (PKC) mediated phosphorylation. We found that phosphorylation of K-Ras at serine 181 partially neutralized the adjacent polybasic stretch of amino acids and thereby activated a farnesyl-electrostatic switch that resulted in release of K-Ras from the plasma membrane and association with intracellular membranes, including the endoplasmic reticulum (ER), Golgi apparatus and the outer mitochondrial membrane. Most intriguing, K-Ras translocation to internal membranes was associated with cell death. Bryostatin 1, a potent PKC agonist, showed anti-tumor activity that was dependent on K-Ras serine 181. In the first cycle of this grant we proposed to expand upon these discoveries with three Aims: 1) Regulation of the farnesyl-electrostatic switch, 2) Mechanisms of phospho-K-Ras mediated apoptosis and 3) The Role of C-terminal phosphorylation of K-Ras in mouse tumor models. Much progress has been made on each aim. Most exciting are our discoveries that K-Ras signals for cell death from the cytoplasmic face of the ER, that Bcl-XL is required for phospho-K-Ras mediated cell death, and that phospho-K-Ras forms a trimolecular complex with Bcl-XL and the IP3 receptor (IP3R) and regulates the calcium channel activity. We have also been successful in constructing a double knock-in mouse that harbors a conditional oncogenic K-Ras allele that lacks the phosphorylation site at amino acid 181 (LSL-K-Ras12D181A). In this competing renewal application we propose to continue our studies with three aims: Aim 1: Regulation of the IP3 Receptor (IP3R) by phospho-K-Ras. We will characterize both structurally (protein-protein interactions) and functionally (electrophysiology) the molecular interactions between phospho-K-Ras, Bcl-XL and IP3R. We will ascertain if phospho-K-Ras alters mitochondrial calcium homeostasis. We will determine if IP3R, calpain and autophagy are required for phospho-K-Ras mediated cell death. Aim 2: Analysis of K-Ras Phosphorylation at Serine 181 in vivo. We will use our newly created LSL-K-Ras12D181A mice in two Cre- driven tumor models to test the hypothesis that phosphorylation at serine 181 negatively regulates K-Ras oncogenicity and we will use the same models to show that the efficacy of bryostatin 1 depends on phosphorylation of serine 181. Aim 3: Analysis of K-Ras Phosphorylation at Serine 181 in Human Tumor Cells. We will correlate susceptibility of human tumor cells lines with K-Ras mutation status and generate isogenic lines of human tumor cells with and without a phosphorylation site at position 181. We anticipate that our mechanistic studies of the cell biology of phospho-K-Ras along with our in vivo and human tumor cell analyses will reveal unique features of this important oncogene that can be exploited in developing anti-cancer drugs. PUBLIC HEALTH RELEVANCE: Oncogenes are genes that cause cancer. K-Ras is the most important human oncogene. We discovered that K-Ras can be modified by the addition of a phosphate group and that this modification inhibits its cancer-promoting activity. We propose to study the cell biology and physiology of K-Ras phosphorylation to better understand how to exploit this process to develop anti-cancer drugs.
Funding Period: 2005-07-01 - 2016-07-31
more information: NIH RePORT

Top Publications

  1. pmc Compartmentalized signaling of Ras in fission yeast
    Brian Onken
    Department of Molecular and Cell Biology, The Breast Center, Baylor College of Medicine, 1 Baylor Plaza, BCM 600, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 103:9045-50. 2006
  2. pmc Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression
    Helen Court
    J Clin Invest 123:4681-94. 2013
  3. pmc Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector
    Joseph P Wynne
    Cancer Institute, NYU School of Medicine, New York, NY 10016, USA
    J Cell Biol 199:317-30. 2012
  4. pmc Regulation of RAS oncogenicity by acetylation
    Moon Hee Yang
    Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA
    Proc Natl Acad Sci U S A 109:10843-8. 2012
  5. pmc Ras hitchhikes on PDE6δ
    Mark R Philips
    NYU Cancer Institute, 522 First Avenue, New York, New York 10016, USA
    Nat Cell Biol 14:128-9. 2012
  6. pmc Regulating the regulator: post-translational modification of RAS
    Ian M Ahearn
    NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA
    Nat Rev Mol Cell Biol 13:39-51. 2012
  7. pmc The perplexing case of the geranylgeranyl transferase-deficient mouse
    Mark R Philips
    NYU Cancer Institute, Smilow 1207, 522 First Avenue, New York, New York 10016, USA
    J Clin Invest 121:510-3. 2011
  8. pmc FKBP12 binds to acylated H-ras and promotes depalmitoylation
    Ian M Ahearn
    Department of Medicine, NYU Langone School of Medicine, 550 First Avenue, New York, NY 10016, USA 0016, USA
    Mol Cell 41:173-85. 2011
  9. pmc Cytosolic Ras supports eye development in Drosophila
    Pamela J Sung
    Department of Medicine, New York University School of Medicine, New York, NY 10016, USA
    Mol Cell Biol 30:5649-57. 2010
  10. pmc Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation
    James P Madigan
    Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
    Biochem J 424:153-61. 2009

Research Grants

  1. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013

Detail Information

Publications18

  1. pmc Compartmentalized signaling of Ras in fission yeast
    Brian Onken
    Department of Molecular and Cell Biology, The Breast Center, Baylor College of Medicine, 1 Baylor Plaza, BCM 600, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 103:9045-50. 2006
    ..These observations provide a striking demonstration of compartment-specific Ras signaling and indicate that spatial specificity in the Ras pathway is evolutionarily conserved...
  2. pmc Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression
    Helen Court
    J Clin Invest 123:4681-94. 2013
    ..Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling...
  3. pmc Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector
    Joseph P Wynne
    Cancer Institute, NYU School of Medicine, New York, NY 10016, USA
    J Cell Biol 199:317-30. 2012
    ..Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2)...
  4. pmc Regulation of RAS oncogenicity by acetylation
    Moon Hee Yang
    Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA
    Proc Natl Acad Sci U S A 109:10843-8. 2012
    ..These data suggest that lysine acetylation is a negative regulatory modification on RAS. Because mutations in RAS family members are extremely common in cancer, modulation of RAS acetylation may constitute a therapeutic approach...
  5. pmc Ras hitchhikes on PDE6δ
    Mark R Philips
    NYU Cancer Institute, 522 First Avenue, New York, New York 10016, USA
    Nat Cell Biol 14:128-9. 2012
    ..PDE6δ is now revealed to be a farnesyl-binding chaperone that facilitates the trafficking and signalling of Ras...
  6. pmc Regulating the regulator: post-translational modification of RAS
    Ian M Ahearn
    NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA
    Nat Rev Mol Cell Biol 13:39-51. 2012
    ....
  7. pmc The perplexing case of the geranylgeranyl transferase-deficient mouse
    Mark R Philips
    NYU Cancer Institute, Smilow 1207, 522 First Avenue, New York, New York 10016, USA
    J Clin Invest 121:510-3. 2011
    ..This surprising result calls into question the role of protein geranylgeranylation in inflammatory cell signaling...
  8. pmc FKBP12 binds to acylated H-ras and promotes depalmitoylation
    Ian M Ahearn
    Department of Medicine, NYU Langone School of Medicine, 550 First Avenue, New York, NY 10016, USA 0016, USA
    Mol Cell 41:173-85. 2011
    ..These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines...
  9. pmc Cytosolic Ras supports eye development in Drosophila
    Pamela J Sung
    Department of Medicine, New York University School of Medicine, New York, NY 10016, USA
    Mol Cell Biol 30:5649-57. 2010
    ..We conclude that the membrane association of Drosophila Ras1 is not required for eye development...
  10. pmc Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation
    James P Madigan
    Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
    Biochem J 424:153-61. 2009
    ..These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization...
  11. pmc Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis
    Roberto J Botelho
    Department of Chemistry and Biology, Ryerson University, Toronto, Ontario M5B 2K3, Canada
    J Biol Chem 284:28522-32. 2009
    ..We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response...
  12. pmc Ras/MAPK signaling from endomembranes
    Nicole Fehrenbacher
    Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA
    Mol Oncol 3:297-307. 2009
    ..Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output...
  13. pmc Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane
    Adam Mor
    The Cancer Institute, NYU School of Medicine, New York, New York 10016, USA
    Mol Cell Biol 29:3297-306. 2009
    ..Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane...
  14. pmc Activation of the MAPK module from different spatial locations generates distinct system outputs
    Kerry Inder
    Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia
    Mol Biol Cell 19:4776-84. 2008
    ....
  15. pmc Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division
    David Michaelson
    Department of Medicine, New York University School of Medicine, New York, NY 10016, USA
    J Cell Biol 181:485-96. 2008
    ..These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division...
  16. pmc Spatial segregation of Ras signaling: new evidence from fission yeast
    Eric C Chang
    Baylor College of Medicine, Department of Molecular and Cell Biology, The Breast Center, Houston, Texas 77030, USA
    Cell Cycle 5:1936-9. 2006
    ..This study has provided unambiguous evidence for compartmentalized signaling of Ras...
  17. pmc Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors
    Pamela J Sung
    New York University NYU Cancer Institute, NYU School of Medicine, New York, NY 10016
    Proc Natl Acad Sci U S A 110:20593-8. 2013
    ..Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors. ..

Research Grants30

  1. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
    ..The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases. ..