Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

James L. Hougland, Katherine A. Hicks, Heather L. Hartman, Rebekah A. Kelly, Terry J. Watt, Carol A. Fierke

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a2 position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca1a2X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a2 and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.

Original languageEnglish (US)
Pages (from-to)176-190
Number of pages15
JournalJournal of Molecular Biology
Volume395
Issue number1
DOIs
StatePublished - Jan 8 2010
Externally publishedYes

Keywords

  • farnesylation
  • peptide library
  • prenylation
  • protein farnesyltransferase
  • substrate specificity

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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