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. 2015 Jul 17;290(36):21845–21856. doi: 10.1074/jbc.M115.676221

FIGURE 4.

FIGURE 4.

ZIP releases the pseudosubstrate of PKCλ from p62 to allow intramolecular autoinhibition of the kinase. A, schematic showing fusion of the PKC activity reporter CKAR to the N terminus of p62. Phosphorylation causes an intramolecular clamp of the phosphorylated segment (pink) with a phospho-peptide-binding FHA2 module (orange) that results in a decrease in FRET between the flanking CFP (blue) and YFP (yellow) (see Ref. 33 for more details). B, COS-7 cells co-expressing CKAR-p62 and mCherry-tagged wild-type PKCλ (black circles, n = 44), PKCλ ΔPS (red squares, n = 19), or PKCι A129E (green triangles, n = 41) were pretreated with 1 μm Gö6983, an inhibitor of conventional and novel PKC isozymes, and then monitored for CFP/FRET ratio changes following 5 μm ZIP treatment. The control CKAR-p62 T/A (blue triangles, n = 23), where its phospho-acceptor Thr was mutated to Ala, was also examined under the same experimental conditions. Data were plotted as PKC activity and represent the normalized FRET/CFP ratio mean ± S.E. of the indicated number of cells from at least 3 independent experiments. C, the FRET/CFP ratio of COS-7 cells co-expressing CFP-p62 and YFP-PKCι (black circles, n = 31) or YFP-PKCι A129E (green diamonds, n = 28) was monitored before and after 5 μm ZIP treatment. Data were plotted as a p62-aPKC interaction and represent the normalized FRET/CFP ratio mean ± S.E. of the indicated number of cells from at least 3 independent experiments. D, COS-7 cells co-expressing CKAR-p62 and mCherry-tagged wild-type PKCλ (black circles, n = 28), PKCλ ΔPS (red squares, n = 30), or PKCι A129E (green triangles, n = 18) were monitored for CFP/FRET ratio changes before and after treatment with 5 μm PZ09, an atypical PKC-selective inhibitor (48). E, COS-7 cells co-expressing CKAR-p62 and either mCherry-tagged wild-type PKCλ (black circles, n = 33) or PKCλ ΔPS (red squares, n = 38) were monitored for CFP/FRET ratio changes when treated with 50 nm calyculin A, a Ser/Thr protein phosphatase inhibitor (56). Data were plotted as PKC activity and represent the normalized FRET/CFP ratio mean ± S.E. of the indicated number of cells from at least 3 independent experiments. F, quantification of basal and phosphatase-suppressed activities of PKCλ and PKCλ ΔPS was calculated as a fraction of FRET ratio change. The data were extrapolated from the plateau portion of the curves in panels D and E, 20 min after the addition of PZ09 or calyculin A.