Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62

J Biol Chem. 2015 Sep 4;290(36):21845-56. doi: 10.1074/jbc.M115.676221. Epub 2015 Jul 17.

Abstract

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.

Keywords: atypical protein kinase C (aPKC); enzyme mechanism; p62 (sequestosome 1(SQSTM1)); protein-protein interaction; scaffold protein; serine/threonine protein kinase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Blotting, Western
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescence Resonance Energy Transfer
  • HEK293 Cells
  • Humans
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Pregnancy Proteins / chemistry
  • Pregnancy Proteins / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Kinase C / chemistry
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Structure, Tertiary
  • Receptors, AMPA / genetics
  • Receptors, AMPA / metabolism
  • Sequestosome-1 Protein
  • Static Electricity

Substances

  • Adaptor Proteins, Signal Transducing
  • Isoenzymes
  • Pregnancy Proteins
  • Receptors, AMPA
  • SQSTM1 protein, human
  • Sequestosome-1 Protein
  • zeta inhibitor protein 14 kDa, human
  • protein kinase C zeta
  • PKC-3 protein
  • Protein Kinase C
  • protein kinase C lambda

Associated data

  • PDB/2KTR