Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62.
Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we ... have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879-12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Blotting, Western, COS Cells, Cercopithecus aethiops, Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Isoenzymes, Models, Molecular, Molecular Sequence Data, Pregnancy Proteins, Protein Binding, Protein Conformation, Protein Kinase C, Protein Structure, Tertiary, Receptors, AMPA, Static Electricity
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Blotting, Western, COS Cells, Cercopithecus aethiops, Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Isoenzymes, Models, Molecular, Molecular Sequence Data, Pregnancy Proteins, Protein Binding, Protein Conformation, Protein Kinase C, Protein Structure, Tertiary, Receptors, AMPA, Static Electricity
J. Biol. Chem.
Date: Sep. 04, 2015
PubMed ID: 26187466
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