Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display.
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.
Wang
B
B
Rammelkamp Center for Research, Case Western Reserve University, Cleveland, Ohio 44109, USA.
Yang
H
H
Liu
Y C
YC
Jelinek
T
T
Zhang
L
L
Ruoslahti
E
E
Fu
H
H
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001667:Binding, Competitive / Q000235:genetics; D017124:Conserved Sequence; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D008024:Ligands; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases