Identification of lysine methylation in the core GTPase domain by GoMADScan

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.

Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

“…It is well-known that the G-domain and C-terminal region of RAS is regulated by various PTMs ( Ahearn et al, 2018 ). Furthermore, accumulating evidence indicates that RAS undergoes S-nitrosylation of select cysteine residues, as well as acetylation, methylation, and ubiquitylation of lysine residues within the G4 and G5 motifs ( Lander et al, 1995 ; Sasaki et al, 2011 ; Knyphausen et al, 2016 ; Yoshino et al, 2019 ; Figure 3 ). These PTMs can upregulate RAS activity by increasing the guanine nucleotide exchange rate and/or inhibiting GAP-mediated GTP hydrolysis.…”

“…In 2019, mass spectrometry analysis of the immunoprecipitated endogenous RAS identified dimethylation at Lys5, adjacent to the G1 motif, as well as monomethylation at Lys147 in H-RAS ( Figure 3 ) ( Yoshino et al, 2019 ). While it is currently unclear whether Lys5 dimethylation is specific for all RAS isotypes, Lys147 is unique to the H-RAS.…”

KRAS Ubiquitination at Lysine 104 Retains Exchange Factor Regulation by Dynamically Modulating the Conformation of the Interface

“…However, less well characterized are PTMs in the core G-domain. These include distinct lysine modifications, such as acetylation, ubiquitination, and most recently methylation ( Ahearn et al., 2011 ; Yoshino et al., 2019 ). Notably, KRAS monoubiquitination at lysine 147 up-regulates RAS activity, signaling, and tumorigenesis ( Baker et al., 2013a ; Sasaki et al., 2011 ).…”

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

“…6 , green triangle , S11 and 2 ). While the specific modified residue(s) could not be site localized using the proteoform fragmentation data collected here because of the high frequency of candidate Lys residues within this region, this PTM could be analogous to HRAS K147me1 ( 58 ). The functional relevance of this monomethylation is unknown but may present an interesting avenue for future investigations.…”