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PLoS One. 2019 Aug 7;14(8):e0219436. doi: 10.1371/journal.pone.0219436. eCollection 2019.

Identification of lysine methylation in the core GTPase domain by GoMADScan.

Author information

1
Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.
2
Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.
3
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.
4
University of North Carolina, R. L. Juliano Structural Bioinformatics Core Facility, Chapel Hill, North Carolina, United States of America.
5
Department of Neurology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.
6
Harvard Medical School, Department of Medicine and Beth Israel Deaconess Medical Center, Division of Signal Transduction, Boston, Massachusetts, United States of America.
7
Department of Human Genetics, University of Michigan, 5815 Medical Science II, Ann Arbor, Michigan, United States of America.
8
Department of Cancer Biology, University of Cincinnati College of Medicine, Ohio, United States of America.
9
Department of Neurosurgery, Brain Tumor Center at UC Gardner Neuroscience Institute, Cincinnati, Ohio, United States of America.
10
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.

Abstract

RAS is the founding member of a superfamily of GTPases and regulates signaling pathways involved in cellular growth control. While recent studies have shown that the activation state of RAS can be controlled by lysine ubiquitylation and acetylation, the existence of lysine methylation of the RAS superfamily GTPases remains unexplored. In contrast to acetylation, methylation does not alter the side chain charge and it has been challenging to deduce its impact on protein structure by conventional amino acid substitutions. Herein, we investigate lysine methylation on RAS and RAS-related GTPases. We developed GoMADScan (Go language-based Modification Associated Database Scanner), a new user-friendly application that scans and extracts posttranslationally modified peptides from databases. The GoMADScan search on PhosphoSitePlus databases identified methylation of conserved lysine residues in the core GTPase domain of RAS superfamily GTPases, including residues corresponding to RAS Lys-5, Lys-16, and Lys-117. To follow up on these observations, we immunoprecipitated endogenous RAS from HEK293T cells, conducted mass spectrometric analysis and found that RAS residues, Lys-5 and Lys-147, undergo dimethylation and monomethylation, respectively. Since mutations of Lys-5 have been found in cancers and RASopathies, we set up molecular dynamics (MD) simulations to assess the putative impact of Lys-5 dimethylation on RAS structure. Results from our MD analyses predict that dimethylation of Lys-5 does not significantly alter RAS conformation, suggesting that Lys-5 methylation may alter existing protein interactions or create a docking site to foster new interactions. Taken together, our findings uncover the existence of lysine methylation as a novel posttranslational modification associated with RAS and the RAS superfamily GTPases, and putative impact of Lys-5 dimethylation on RAS structure.

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