Isolation of High-Affinity Peptide Antagonists of 14-3-3 Proteins by Phage Display

Biochemistry. 1999 Sep 21;38(38):12499-504. doi: 10.1021/bi991353h.

Abstract

The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K(D) values of 7-9 x 10(-)(8) M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3-R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3zeta revealed the occupancy of the general binding groove of 14-3-3zeta by R18, explaining the potent inhibitory effect of R18 on 14-3-3-ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 14-3-3 Proteins
  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Bacteriophages / genetics*
  • Binding, Competitive / genetics
  • Conserved Sequence
  • Humans
  • Ligands
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Peptides / genetics
  • Peptides / isolation & purification*
  • Peptides / metabolism*
  • Phosphoprotein Phosphatases / metabolism
  • Protein Binding / genetics
  • Protein Isoforms / genetics
  • Protein Isoforms / isolation & purification
  • Protein Isoforms / metabolism
  • Proteins / antagonists & inhibitors*
  • Proteins / genetics
  • Proteins / metabolism
  • Proto-Oncogene Proteins c-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins c-raf / metabolism
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • Ligands
  • Peptides
  • Protein Isoforms
  • Proteins
  • Tyrosine 3-Monooxygenase
  • Proto-Oncogene Proteins c-raf
  • Phosphoprotein Phosphatases