Ras proteins regulate multiple cellular signaling pathways through interaction with an array of effectors including Raf kinases, phosphatidylinositol-3-kinase, Ral GEFs, and Nore1. The best characterized of these regulatory interactions occur on the cytoplasmic surface of the plasma membrane in response to signals from growth factor receptors. However, it is now apparent that Ras also signals from endomembrane compartments and distinct microdomains of the plasma membrane. This has heightened interest in defining the mechanisms of subcellular targeting of Ras and other small monomeric GTPases. We have focused on the subcellular targeting of Ras proteins as a model to understand how lipid modification contributes to protein trafficking and signaling. Ras proteins are posttranslationally modified by CaaX box prenylation (farnesylation), -aaX cleavage, methylation, and with the exception of K-Ras, palmitoylation. Posttranslational modification is required for subcellular targeting, but also plays a key role in the assembly of Ras-dependent signaling complexes in a spatially and temporally restrictive fashion. Palmitoylation is unique among lipid modifications in that it is readily reversible, controlled by the action of protein acyl transferases (DHHC PATs) and proposed acyl protein thioesterases (APTs). A model for Ras trafficking has been proposed that is based on cycles of acylation and deacylation coupled with membrane exchange (kinetic trap). The current proposal focuses on palmitoylation and depalmitoylation of yeast Ras proteins and determines how acylation/deacylation cycles control trafficking and signaling. Mutations in ras genes have been implicated in approximately 30% of all cancers, with the incidence Ras mutations appearing in some cancers being considerably higher. We have discovered that lipid modification of Ras proteins is required for Ras function and propose to study the role of lipid modification as a potential target of cancer chemotherapeutic drugs. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM073977-02
Application #
7803462
Study Section
Cell Structure and Function (CSF)
Program Officer
Shapiro, Bert I
Project Start
2008-03-01
Project End
2012-02-29
Budget Start
2009-01-16
Budget End
2009-02-28
Support Year
2
Fiscal Year
2008
Total Cost
$44,243
Indirect Cost
Name
University of South Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Hamel, Laura D; Deschenes, Robert J; Mitchell, David A (2014) A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening. Anal Biochem 460:1-8
Mitchell, David A; Hamel, Laura D; Reddy, Krishna D et al. (2014) Mutations in the X-linked intellectual disability gene, zDHHC9, alter autopalmitoylation activity by distinct mechanisms. J Biol Chem 289:18582-92
Mitchell, David A; Mitchell, Gayatri; Ling, Yiping et al. (2010) Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes. J Biol Chem 285:38104-14
Vinnakota, Kalyan C; Mitchell, David A; Deschenes, Robert J et al. (2010) Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching. Phys Biol 7:026011
Jennings, Benjamin C; Nadolski, Marissa J; Ling, Yiping et al. (2009) 2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro. J Lipid Res 50:233-42