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

J Biol Chem. 2014 Feb 14;289(7):3950-9. doi: 10.1074/jbc.M113.531178. Epub 2013 Dec 13.

Abstract

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.

Keywords: Cancer Biology; Cytokinesis; Dictyostelium; Molecular Cell Biology; Ras; Signaling; Ubiquitination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytokinesis / physiology*
  • Dictyostelium / enzymology*
  • Dictyostelium / genetics
  • Guanosine Triphosphate / genetics
  • Guanosine Triphosphate / metabolism
  • Humans
  • Lysine / genetics
  • Lysine / metabolism
  • Protein Stability
  • Proteolysis*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins p21(ras)
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination / physiology*
  • ras Proteins / genetics
  • ras Proteins / metabolism*

Substances

  • KRAS protein, human
  • Proto-Oncogene Proteins
  • Protozoan Proteins
  • rasG protein, Dictyostelium
  • Guanosine Triphosphate
  • Ubiquitin-Protein Ligases
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins
  • Lysine