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Scaffolding of atypical protein kinase C to p62 is disrupted by the pseudosubstrate peptide ZIP (802.4)
Author(s) -
Tsai LiChun,
Xie Lei,
Xie Li,
Bourne Philip,
Newton Alexandra
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.802.4
Subject(s) - förster resonance energy transfer , peptide , protein–protein interaction , scaffold protein , docking (animal) , biophysics , myristoylation , protein kinase c , microbiology and biotechnology , chemistry , biochemistry , protein kinase a , plasma protein binding , binding site , biology , kinase , fluorescence , signal transduction , phosphorylation , medicine , physics , nursing , quantum mechanics
Atypical protein kinase C (aPKC) enzymes, aPKCλ and aPKCζ, play key roles in metabolic signaling. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity, however we have recently shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu‐Zhang et al., (2012) JBC 287:12879‐85). Here, we identify a novel interaction between the pseudosubstrate segment of PKCλ and the protein scaffold p62, a known binding partner, that is disrupted by ZIP in cells. First, we used protein‐protein interaction network analysis, structural modeling, and protein‐protein docking to predict that ZIP, a basic peptide, binds an acidic surface on the PB1 domain of p62. This predicted interaction is additional to the known interaction between the PB1 domain of aPKC, which immediately precedes the pseudosubstrate segment, and p62. Second, we used fluorescence resonance energy transfer (FRET) experiments to show that ZIP disrupts the interaction of CFP‐tagged p62 and YFP‐tagged PKCλ in cells via a mechanism that depends on an intact pseudosubstrate segment. In contrast, auranofin, a known disrupter of the PB1:PB1 domain interaction of aPKC and p62, disrupts the interaction of wild‐type PKCλ, a construct lacking the pseudosubstrate, but not a construct in which the PB1 domain is deleted. Our data are consistent with ZIP altering cellular function by displacing binding partners from the acidic face of the PB1 domain of p62, a region which we identify as an interaction surface for the pseudosubstrate segment of aPKCs. Grant Funding Source : This work was supported by 5T32DK007494 and P01 DK054441.