Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs

Amanda S. Varner, Charles E. Ducker, Zuping Xia, Yan Zhuang, Mackenzie L. De Vos, Charles D. Smith

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity, towards the myristoylated substrate peptide but not the farnesylated peptide, while ras-transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the Km values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the Km for MyrGCK(NBD) was 5-fold lower than the Km for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC50 values of approx. 4 and 1.3 μM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N-ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of famesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist.

Original languageEnglish (US)
Pages (from-to)91-99
Number of pages9
JournalBiochemical Journal
Volume373
Issue number1
DOIs
StatePublished - Jul 1 2003

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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