The goal of this work is to understand the function of the ras oncogene protein in signal transduction and the control of cell growth. Mutational activation of the ras oncogene is involved in more that 25% of all human malignancies and is, therefore, a major factor in tumor formation. RAS proteins are small (21-38 kD), GTP binding proteins that are highly conserved in evolution. Members of the RAS family have been identified in every eucaryote examined from mammals to the yeast Saccharomyces cerevisiae. In every system examined, the role of RAS proteins in the control of cell growth requires membrane localization. Yet, RAS is synthesized initially as a soluble protein and membrane localization requires a series of posttranslational modifications. The steps involved in RAS processing include proteolytic cleavage, carboxymethylation, isoprenylation and palmitoylation. This proposal described experiments directed at characterization of these activities and to assess the role of these modifications in the control of RAS activity. The system that has been chosen in which to carry out this work is the yeast Saccharomyces cerevisiae. The use of yeast allows a powerful combination of experimental approaches to be applied to the problem. Specifically, experiments will be described that (1) Construct mutants in the C terminus of yeast RAS protein to define the structural determinants of the RAS localization signal (2) Characterize the enzymatic steps responsible for maturation of RAS (3) Examine the role of RAS modifications on the control of yeast cell growth, and (4) Carry out genetic selections to isolate mutants in the RAS processing pathway. Since membrane localization is required for RAS function, the cell may use these steps to control RAS activity. Therefore, inhibitors of specific steps in the RAS maturation pathway may be effective targets for the design of therapeutic agents to inhibit RAS transformed cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Physiological Chemistry Study Section (PC)
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University of Iowa
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Iowa City
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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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