Abstract Members of the Ras superfamily of small GTPases act as binary switches that regulate numerous biological pathways, including cell proliferation, survival, and motility. Activating mutations in Ras genes are oncogenic and are found in more than 30% of human cancers. Rho GTPases play important roles in tumorigenesis and metastasis. Accordingly, these Ras-family GTPases are targets for therapeutic intervention in cancer. Two protein posttranslational lipid modifications are well known to regulate Ras GTPases, cysteine prenylation and cysteine palmitoylation. Our recent studies have identified a novel posttranslational modification (PTM), reversible lysine palmitoylation, which regulates KRas4A, a KRAS splice variant that is broadly expressed in cancer cell lines and human colorectal tumors. We have identified an enzyme that removes lysine palmitoylation from this protein, resulting in an increase in KRas4A transforming activity. Based on this exciting discovery, the specific aims of the project are (1) to determine whether other small GTPases are also regulated by reversible lysine palmitoylation. Bioorthogonal click chemistry will be used to detect lysine palmitoylation and its regulation by sirtuins will be established using knockdown or knockout strategies in mammalian cells. (2) To identify the mechanism of palmitate addition to lysine residues in GTPases, we will use two approaches to establish a role for DHHC palmitoyltransferases. First, we will examine how lysine palmitoylation levels change when DHHC enzymes are overexpressed or knocked down in mammalian cells. Second, we will reconstitute lysine palmitoylation in vitro using purified Ras proteins and DHHC enzymes. (3) To determine how lysine palmitoylation regulates GTPase activity, we will assess the impact of lysine palmitoylation on assays of Ras biological activity and its interactions with regulators and effectors. Our proposed studies will establish a novel regulatory mechanism for the Ras family of small GTPases and provide interesting new insights into their biological function. In addition, our studies will for the first time establish the biological significance of a previously under-recognized PTM, lysine palmitoylation. Because Ras-family proteins are considered important therapeutic targets, our studies have the potential to identify new strategies for cancer treatment.