MembersoftheRassuperfamilyofsmallGTPasesactasbinaryswitchesthatregulatenumerousbiological pathways,includingcellproliferation,survival,andmotility.ActivatingmutationsinRasgenesareoncogenic 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),reversiblelysinepalmitoylation,whichregulatesKRas4A,aKRASsplicevariantthatis 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 GTPasesarealsoregulatedbyreversiblelysinepalmitoylation.Bioorthogonalclickchemistrywillbeusedto 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 proteinsandDHHCenzymes.(3)TodeterminehowlysinepalmitoylationregulatesGTPaseactivity,wewill 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, lysinepalmitoylation.BecauseRas-familyproteinsareconsideredimportanttherapeutictargets,ourstudies havethepotentialtoidentifynewstrategiesforcancertreatment.

Public Health Relevance

Ras mutations are frequently found in human tumors and are considered important targets for cancer therapy. Efforts to date have not yielded successful therapeutic targeting of Ras oncoproteins. Our proposedstudywillelucidateanovelregulatorymechanismforRas-familyGTPases,significantlyimproving our understanding of their biology and uncovering new ways to target Ras-family proteins for cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM121540-03
Application #
9731578
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Barski, Oleg
Project Start
2017-07-15
Project End
2021-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Cornell University
Department
Other Basic Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Zhang, Xiaoyu; Cao, Ji; Miller, Seth P et al. (2018) Comparative Nucleotide-Dependent Interactome Analysis Reveals Shared and Differential Properties of KRas4a and KRas4b. ACS Cent Sci 4:71-80
Jing, Hui; Zhang, Xiaoyu; Wisner, Stephanie A et al. (2017) SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a. Elife 6: