Mutation-specific and common phosphotyrosine signatures of KRAS G12D and G13D alleles
Embargo until
2019-12-01
Date
2018-08-01
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Johns Hopkins University
Abstract
KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutations, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as non-receptor tyrosine kinases, MAPK kinases and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect the subtle differences between related oncogenic mutants and potentially lead to individualized treatments.
Our findings from studies focusing on two different oncogenic mutations of KRAS highlighted the common hyperphosphorylation of TNK2, a non-receptor tyrosine kinase that is known to be mutated, overexpressed, or hyperphosphorylated in several cancer subtypes. Non-receptor tyrosine kinases represent an important class of signaling molecules which are involved in driving diverse cellular pathways. Although the large majority have been well-studied in terms of their protein binding partners, the interactomes of some important non-receptor tyrosine kinases such as TNK2 (also known as activated Cdc42-associated kinase 1 or ACK1) have not been systematically investigated. Aberrant expression and hyperphosphorylation of TNK2 have been implicated in a number of cancers, although the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent protein labeling methods, such as biotinylation identification (BioID), are being increasingly used to map protein-protein interactomes as they provide increased sensitivity in finding interacting proteins. Therefore, as an extension of our effort to understand oncogenic KRAS signaling and its components, we sought to map the interacting partners of TNK2. We also employed Stable Isotope Amino Acid Labeling in Cell Culture (SILAC) to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were not only able confidently identify site-level biotinylation of previously well-established TNK2 binders and substrates (NCK1, NCK2, CTTN, STAT3), but also identify of several novel TNK2 interactors. We validated TNK2 interaction with one novel interactors, clathrin interactor 1 (CLINT1), using immunoblot analysis. Overall, this work highlights several molecules that warrant further experiments to assess their functional significance in TNK2-mediated signaling.
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Keywords
KRAS, proteomics, mass spectrometry, cancer signaling, protein-protein interaction, kinases