Abstract

The long-term goal of the proposed research is to understand how modification of proteins by myristylation affects protein structure and function. This will be accomplished by studying N-myristyl transferase (NMT), the enzyme which catalyzes attachment of myristate to proteins, as well as its protein substrates. NMT will be purified from mammalian liver, and used as an antigen for production of polyclonal and monoclonal antisera. Anti-NMT antibodies will be employed, in conjunction with NMT activity assays, to probe the subcellular localization of NMT in normal and Rous sarcoma virus-transformed cells. In addition, the gene encoding NMT will be cloned and used to express NMT. Cellular substrates for NMT will be identified by immunoreaction with anti-myristylglycine antibodies, and effects of oncogenic transformation on the expression and distribution of myristylated proteins will be studied. Additional experiments will focus on the myristylated transforming protein of Rous sarcoma virus, pp60(v-src), which requires myristylation in order to bind to membranes and elicit transformation. The effect of altering fatty acid hydrophobicity, chain length, and protein sequence context of the myristate moiety on pp60(v-src) will be tested in an in vitro membrane binding assay. Since myristate is attached to numerous viral structural proteins and oncogenes, as well as to proteins involved in growth control and signal transduction, the proposed studies should aid in understanding the basic biochemical mechanisms which regulate cell growth.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA052405-03
Application #
3197117
Study Section
Biochemistry Study Section (BIO)
Project Start
1990-03-01
Project End
1995-02-28
Budget Start
1992-03-01
Budget End
1993-02-28
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Saouaf, S J; Wolven, A; Resh, M D et al. (1997) Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate. Biochem Biophys Res Commun 234:325-9
Parent, L J; Wilson, C B; Resh, M D et al. (1996) Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein. J Virol 70:1016-26
Berthiaume, L; Resh, M D (1995) Biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins. J Biol Chem 270:22399-405
Berthiaume, L; Peseckis, S M; Resh, M D (1995) Synthesis and use of iodo-fatty acid analogs. Methods Enzymol 250:454-66
Berthiaume, L; Deichaite, I; Peseckis, S et al. (1994) Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins. J Biol Chem 269:6498-505
Sigal, C T; Zhou, W; Buser, C A et al. (1994) Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids. Proc Natl Acad Sci U S A 91:12253-7
Alland, L; Peseckis, S M; Atherton, R E et al. (1994) Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J Biol Chem 269:16701-5
Peseckis, S M; Resh, M D (1994) Fatty acyl transfer by human N-myristyl transferase is dependent upon conserved cysteine and histidine residues. J Biol Chem 269:30888-92
Zhou, W; Parent, L J; Wills, J W et al. (1994) Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol 68:2556-69
Buser, C A; Sigal, C T; Resh, M D et al. (1994) Membrane binding of myristylated peptides corresponding to the NH2 terminus of Src. Biochemistry 33:13093-101

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