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

Abstract

The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKCepsilon) by engaging two tandem phosphoserine-containing motifs located between the PKCepsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKCepsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3zeta bound to a synthetic diphosphorylated PKCepsilon 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 PKCepsilon. 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.

Figure 1

Stoichiometry and thermodynamics of the 14-3-3–PKCɛ interaction. (A) Schematic showing PKCɛ primary structure, 14-3-3 phosphoserine-binding motifs and idealized 14-3-3 consensus sequences underneath. (B) The 14-3-3ζ–GFP-PKCɛ complex migrates by size exclusion chromatography with an apparent molecular weight that is consistent with a stoichiometry of one GFP–PKCɛ monomer (110 kDa) to one 14-3-3ζ dimer (58 kDa). (C) Isothermal titration calorimetry experiments showing binding curves for PKCɛV3 monophosphopeptides and diphosphopeptide. Raw data are shown in the top panels with fitted curves below. GFP, green fluorescent protein, PKCɛ, protein kinase C-ɛ.

Figure 2

Structure of the 14-3-3–PKCɛV3 complex. (A) The electron density (2mFo-DFc contoured at σ=1.2) for the 14-3-3–PKCɛV3-pS346-pS368 diphosphopeptide complex is shown. (B) Surface representation of the 14-3-3 dimer (protomers are shown in blue and green, respectively) with the PKCɛV3-pS346-pS368 peptide shown as sticks in yellow, and the location and distance spanned by the missing connecting linker indicated. The figure was prepared using PyMOL (Delano, 2002). PKCɛ, protein kinase C-ɛ.

Figure 3

Close-up of the molecular contacts made by each phosphoserine binding site and structural comparisons. (A) Hydrogen bonds are shown for site 1 (left panel) and site 2 (right panel) with selected interaction residues labelled in pink (14-3-3) and black (PKCɛ). (B) Comparison of the PKCɛV3 site 1 with a mode 1 14-3-3 peptide (left panel, grey, PDB code 1QJB) and site 2 with a mode 2 peptide (right panel, grey, PDB code 1QJA). PKCɛV3-pS346-pS368 is shown in yellow, 14-3-3 chain A is green and chain B is blue. Residue numbers are labelled as in panel (A). The figure was prepared using PyMOL (Delano, 2002). PDB, protein data bank; PKCɛ, protein kinase C-ɛ.

Figure 4

Distinct binding modes for 14-3-3 partner proteins and selected examples of proteins with known two-site intra-chain 14-3-3 binding sites. (A) Structures of inter-chain (symmetric) complexes with a 14-3-3 dimer (blue ribbon) compared with the intra-chain (asymmetric) structure described for the PKCɛV3 ligand. (B) The domain structure and location of consensus and divergent 14-3-3 sites within selected intra-chain two-site 14-3-3 partners. Optimal motifs are defined as those containing at least three out of four consensus mode 1 or mode 2 residues (Peng et al, 1997; Obsil et al, 2003; Ganguly et al, 2005; Kim et al, 2007; Saurin et al, 2008). PDB, protein data bank; PKCɛ, protein kinase C-ɛ.