Understanding the function of the Ras oncogene at the molecular level is a high priority of cancer research because mutational activation of Ras is involved in large number of neoplasias. Ras proteins are low molecular weight GTP binding proteins that function as on/off switches to regulate signal transduction pathways. Ras is active when in the GTP bound state. Hydrolysis of GTP to GDP inactivates Ras as a signaling molecule. In addition to the regulation of Ras by guanine nucleotides, Ras function requires a series of posttranslational modifications. Work accomplished during the first funding period of this grant elucidated the posttranslational events required to generate functional Ras protein. The steps include; (1) farnesylation of a cysteine that occurs in a conserved C-terminal sequence motif referred to as the CaaX box (C is Cys, a is any aliphatic residue, and X is the C-terminal residue), (2) proteolytic removal of the -aaX sequence, (3) carboxyl methylation of the newly exposed cysteinyl carboxyl, and (4) palmitoylation of a second cysteine residue often found close to the CaaX box. One function of these modifications is to redirect Ras from the cytoplasm to the plasma membrane where it interacts with cellular effectors to modulate signal transduction pathways. The goal of this project is to determine how posttranslational events regulate the function of Ras. The work described in this proposal will focus on the Ras gene homologs found in Saccharomyces cerevisiae. The yeast system has been chosen so that a combination of biochemical and genetic techniques can be employed. Ras mutants will be constructed and their activity measured by in vivo and in vitro techniques. In work leading up to this proposal we have found that addition of the palmitoyl moiety to Ras plays an important role in directing Ras to its site of action in the plasma membrane. In the current study, we propose methods to isolate the palmitoyl transferase gene and to study the biochemical activity of the palmitoyl transferase enzyme. Although the mechanism by which CaaX box processing controls Ras function is still unclear, inhibitors of the Ras processing pathway are being developed as potential cancer chemotherapeutic agents. The experiments we propose will improve our understanding of Ras function and will contribute to efforts to design drugs that interfere with the activity of activated Ras alleles.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA050211-09
Application #
2712630
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Spalholz, Barbara A
Project Start
1990-08-01
Project End
2000-02-29
Budget Start
1998-06-01
Budget End
2000-02-29
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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; Hamel, Laura D; Ishizuka, Kayoko et al. (2012) The Erf4 subunit of the yeast Ras palmitoyl acyltransferase is required for stability of the Acyl-Erf2 intermediate and palmitoyl transfer to a Ras2 substrate. J Biol Chem 287:34337-48
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
Linder, Maurine E; Deschenes, Robert J (2007) Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8:74-84
Mitchell, David A; Vasudevan, Anant; Linder, Maurine E et al. (2006) Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res 47:1118-27
Budde, Cheryl; Schoenfish, Marissa J; Linder, Maurine E et al. (2006) Purification and characterization of recombinant protein acyltransferases. Methods 40:143-50
Wang, Geng; Deschenes, Robert J (2006) Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae. Mol Cell Biol 26:3243-55

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