12Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many 13 human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling 14 pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras 15 splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-16 translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide 17 (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further 18 demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-19 Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study 20 identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of 21GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological 22 significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. 23 24 25. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx

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