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

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.

Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

“…The ability of 14-3-3 to bind nonphosphorylated peptides and proteins has been demonstrated by several groups; however, the physiological relevance of this potential has not been determined (Petosa et al, 1998;Masters et al, 1999;Wang et al, 1999). Our finding showing that the in vivo 14-3-3-associated proteins in COS-7 cells bind 14-3-3 through the phosphopeptide binding groove, as demonstrated by the ability of a 14-3-3 binding phosphopeptide to displace all specifically associated proteins, combined with the finding that most of these associated proteins are reactive with the pan phospho-specific 14-3-3 binding site antibody, raises questions about the physiological significance of 14-3-3 binding to nonphosphorylated targets.…”

“…Nevertheless, several reports indicate that 14-3-3 proteins can also bind nonphosphorylated peptides and proteins through their phosphopeptide binding pocket (Masters et al, 1999;Wang et al, 1999;Mils et al, 2000). To examine what fraction of the 14-3-3-associated proteins in COS-7 cells contains phosphorylation sites recognized by the pan phosphospecific 14-3-3 binding site antibody, we compared the pattern of 14-3-3-associated proteins detected by metabolic 35 SMet labeling (Figure 2, top) with that detected by immunoblotting with the pan phospho-specific antibody (Figure 2, bottom).…”

14-3-3 Proteins Regulate Protein Kinase A Activity to Modulate Growth Cone Turning Responses

“…R18 (PHCVPRDLSWLDLEANMCLP) is a potent 14-3-3 peptide antagonist, which disrupts binding of Ser/Thr phosphorylated proteins to 14-3-3 (Wang et al, 1999). We generated a recombinant R18-green fluorescent protein expressing herpes simplex virus (HSV-R18-GFP) and a control HSV-WLRL-GFP virus that does not bind to 14-3-3 because of a substitution in the core-binding motif (WLDL to WLRL) (Wang et al, 1999). R18-GFP binds to 14-3-3 and antagonizes a previously characterized 14-3-3 -Slingshot1 interaction (supplemental Fig.…”

Identification of a host 14-3-3 protein that interacts with Xanthomonas effector AvrRxv

“…Analysis of the AvrRxv sequence indicated that there are two domains in AvrRxv that resemble known 14-3-3 binding motifs, RSFDTP (called Raf domain) and MDIE (called R18 domain) [42,45]. To test if either the Raf or R18 domain is important for the AvrRxv and ARI1 interaction, mutant versions of AvrRxv were assayed for interaction with ARI1 in yeast.…”

“…This motif is called the Raf motif because it was first identified in the Raf-1 protein kinase. In addition, there is a 14-3-3 binding motif that does not require phosphorylation called the R18 motif, found in an inhibitory peptide (DI/L/VE) [42]. Although the vast majority of 14-3-3 interactions require phosphorylation, it is not required for binding of 14-3-3 proteins to several cellular proteins in mammals, most importantly, the ADP-ribosyltransferase effector ExoS of Pseudomonas aeruginosa [43][44][45][46].…”