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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 16; pp. 4140 - 4145
• Main Authors: Ntai, Ioanna, Fornelli, Luca, DeHart, Caroline J., Hutton, Josiah E., Doubleday, Peter F., LeDuc, Richard D., van Nispen, Alexandra J., Fellers, Ryan T., Whiteley, Gordon, Boja, Emily S., Rodriguez, Henry, Kelleher, Neil L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 17.04.2018
• Subjects: Amino Acid Sequence; Biological Sciences; Cancer; Carboxymethylation; Cell Line, Tumor; Cell Membrane - metabolism; Cell survival; Cells; Chromatography, Liquid; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - enzymology; Colorectal Neoplasms - genetics; Crosstalk; Cysteine - chemistry; Genes; Genotypes; Humans; Immunology; K-Ras protein; Mass spectrometry; Mass spectroscopy; Methylation; Models, Molecular; Mutation; Mutation, Missense; Neoplasm Proteins - analysis; Neoplasm Proteins - chemistry; Neoplasm Proteins - isolation & purification; Nitrosation; Physical Sciences; Point Mutation; Prenylation; Protein Conformation; Protein Processing, Post-Translational; Proteins; Proteomics; 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; Tumor cell lines; Tumorigenesis; Tumors; Workflow; KRAS; RAS; top-down proteomics; posttranslational modification; immunoprecipitation
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1716122115

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.