Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk

Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4140-4145. doi: 10.1073/pnas.1716122115. Epub 2018 Apr 2.

Abstract

Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

Keywords: KRAS; RAS; immunoprecipitation; posttranslational modification; top-down proteomics.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Cell Membrane / metabolism
  • Chromatography, Liquid
  • Colorectal Neoplasms / enzymology*
  • Colorectal Neoplasms / genetics
  • Cysteine / chemistry
  • Humans
  • Methylation
  • Models, Molecular
  • Mutation, Missense*
  • Neoplasm Proteins / analysis*
  • Neoplasm Proteins / chemistry
  • Neoplasm Proteins / isolation & purification
  • Nitrosation
  • Point Mutation*
  • Prenylation
  • Protein Conformation
  • Protein Processing, Post-Translational*
  • Proteomics / methods
  • Proto-Oncogene Proteins p21(ras) / analysis*
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / isolation & purification
  • Recombinant Proteins / chemistry
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Tandem Mass Spectrometry

Substances

  • KRAS protein, human
  • Neoplasm Proteins
  • Recombinant Proteins
  • Proto-Oncogene Proteins p21(ras)
  • Cysteine