title: Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62.
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.
authors.affiliation: From the Department of Pharmacology.
authors.forename: Li-Chun Lisa
authors.initials: LC
authors.lastname: Tsai
authors.affiliation: the Department of Computer Science, Hunter College, the City University of New York, New York, New York 10065.
authors.forename: Lei
authors.initials: L
authors.lastname: Xie
authors.affiliation: Department of Neurosciences.
authors.forename: Kim
authors.initials: K
authors.lastname: Dore
authors.affiliation: Skaggs School of Pharmacy, and.
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.affiliation: From the Department of Pharmacology.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Del Rio
authors.affiliation: Pediatric Diabetes Research Center, University of California San Diego, La Jolla, California 92093.
authors.forename: Charles C
authors.initials: CC
authors.lastname: King
authors.affiliation: the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, and.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Martinez-Ariza
authors.affiliation: the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hulme
authors.affiliation: Department of Neurosciences.
authors.forename: Roberto
authors.initials: R
authors.lastname: Malinow
authors.affiliation: the Office of the Director, the National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Bourne
authors.affiliation: From the Department of Pharmacology, anewton@ucsd.edu.
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D055672:Static Electricity; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D007527:Isoenzymes; D011257:Pregnancy Proteins; D011493:Protein Kinase C; D018091:Receptors, AMPA; D000071456:Sequestosome-1 Protein; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C099718:SQSTM1 protein, human; C510332:zeta inhibitor protein 14 kDa, human; C115015:PKC-3 protein
keywords: atypical protein kinase C (aPKC); enzyme mechanism; p62 (sequestosome 1(SQSTM1)); protein-protein interaction; scaffold protein; serine/threonine protein kinase