Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling

Lauren M. Adams, Caroline Jane DeHart, Bryon S. Drown, Lissa C. Anderson, William Bocik, Emily S. Boja, Tara M. Hiltke, Christopher L. Hendrickson, Henry Rodriguez, Michael Caldwell, Reza Vafabakhsh, Neil L Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top–down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.

Original languageEnglish (US)
Article number102768
JournalJournal of Biological Chemistry
Volume299
Issue number1
DOIs
StatePublished - Jan 2023

Funding

This work was supported by federal funds from the NCI (Office of Cancer Clinical Proteomics Research), National Institutes of Health (NIH), and Leidos Biomedical Research under contract HHSN261200800001E and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under the NIH grant P41 GM108569. A portion of this work was performed at the National High Magnetic Field Laboratory, which is supported by the National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida. We thank Byoung-Kyu and Young ah Goo for their work on BU MS in this study. BU analysis was performed by the Northwestern Proteomics Core Facility, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from the NIH Office of the Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569. We also thank Paul M. Thomas for helpful discussions throughout this work and Karla Satchell for valuable feedback. L. M. A. C. J. D. and N. L. K. conceptualization; L. M. A. C. J. D. L. C. A. and R. V. methodology; L. M. A. C. J. D. and L. C. A. validation; L. M. A. C. J. D. and B. S. D. formal analysis; L. M. A. C. J. D. L. C. A. and R. V. investigation; E. S. B. T. M. H. C. L. H. H. R. R. V. and N. L. K. resources; L. M. A. C. J. D. and L. C. A. data curation; L. M. A. writing–original draft; L. M. A. C. J. D. B. S. D. L. C. A. and N. L. K. writing–review & editing; L. M. A. C. J. D. and M. C. visualization; W. B. C. L. H. and N. L. K. supervision; W. B. E. S. B. T. M. H. C. L. H. H. R. and N. L. K. project administration; W. B. C. L. H. and N. L. K. funding acquisition. L. M. A. was supported by T32GM008382. B. S. D. was supported by the NCI of the NIH under award number F32CA246894. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. L. M. A. was supported by T32GM008382. B. S. D. was supported by the NCI of the NIH under award number F32CA246894. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was supported by federal funds from the NCI (Office of Cancer Clinical Proteomics Research), National Institutes of Health (NIH), and Leidos Biomedical Research under contract HHSN261200800001E and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under the NIH grant P41 GM108569. A portion of this work was performed at the National High Magnetic Field Laboratory , which is supported by the National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida. We thank Byoung-Kyu and Young ah Goo for their work on BU MS in this study. BU analysis was performed by the Northwestern Proteomics Core Facility, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center , instrumentation award ( S10OD025194 ) from the NIH Office of the Director , and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569 . We also thank Paul M. Thomas for helpful discussions throughout this work and Karla Satchell for valuable feedback.

Keywords

  • KRAS
  • MAPK pathway
  • RAS protein
  • colorectal cancer
  • immunoprecipitation
  • post-translational modification
  • proteoform
  • top–down proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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