Recognition of an intra-chain tandem 14-3-3 binding site within PKC epsilon

The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKC epsilon) by engaging two tandem phosphoserine-containing motifs located between the PKC epsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKC epsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3 zeta bound to a synthetic diphosphorylated PKC epsilon V3 region revealing how a consensus 14-3-3 site and a divergent 14-3-3 site cooperate to bind to 14-3-3 and so activate PKC epsilon. Thermodynamic data show a markedly enhanced binding affinity for two-site phosphopeptides over single-site 14-3-3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14-3-3 ligand. This dual-site intra-chain recognition has implications for other 14-3-3 targets, which seem to have only a single 14-3-3 motif, as other lower affinity and cryptic 14-3-3 gatekeeper sites might exist.