Figure 4. Autoinhibition Protects PKC from PHLPP1-Mediated Dephosphorylation and Degradation.
(A) Schematic of indicated PKCβII truncation mutants.
(B) IB analysis of COS7 cells expressing indicated PKCβII constructs probed with indicated phospho-specific or total PKC antibodies.
(C) Autoradiogram (35S) and IB analysis of newly-synthesized PKC pulse-chase immunoprecipitates from COS7 cells expressing HA-PKCβII and FLAG-PHLPP1 (see Methods).
(D) IB analysis of FLAG immunoprecipitates from COS7 cells transfected with indicated HA-PKCβII and FLAG-PHLPP1 constructs and probed with indicated antibodies. Vinculin was used as a loading control.
(E) IB analysis of lysates from COS7 cells transfected with indicated HA-PKCβII constructs probed with phospho-specific or total PKC antibodies. PKCβII Δ37-86 (ΔC1A), PKCβII Δ159-291 (ΔC2), PKCβII Δ101-291 (ΔC1B/C2), PKCβII Δ37-291 (ΔC1A/C1B/C2), or PKCβII 296-673 (Cat). Quantification (bottom) of pSer660band intensity relative to WT (mean ± range, n=2).
(F) COS7 cells co-expressing CKAR and indicated mCherry-PKCβII regulatory domain deletion constructs were treated with BisIV (2μM). Insert shows trace for Cat activity, with cluster of traces in main figure reproduced for comparison. Quantification (bottom) shows magnitude of the FRET ratio change from 3 independent experiments.
(G) Autoradiogram (35S) of HA immunoprecipitates from pulse-chase of COS7 cells expressing the indicated PKC constructs.
(H) IB analysis of lysates from Sf9 insect cells infected with the indicated GST-PKC constructs and His-Phlpp1 PP2C (PHLPP1; 1154-1422) baculovirus, probed with the indicated phospho-specific or total PKC antibodies. Quantification (right) of total PKC protein normalized to Tubulin or PKC phosphorylation normalized to total PKC.
(I) IB analysis of lysates from COS7 cells expressing the indicated HA-PKC constructs treated with cycloheximide (CHX, 250μM) for the indicated times prior to lysis and probed with the indicated antibodies. Quantification (bottom) of PKC band intensity normalized to Tubulin loading control and plotted as percentage of protein at time zero.
(J) IB analysis of lysates from WT (Phlpp1+/+) or PHLPP1 knock-out (Phlpp1−/−) MEFs treated with PDBu (200 nM) for the indicated time points prior to lysis and probed with the indicated antibodies. Quantification (bottom) of PKC band intensity normalized to Hsp90 loading control and plotted as percentage of protein at time zero.
(K) IB analysis of lysates from untreated WT (Phlpp1+/+) or PHLPP1 knock-out (Phlpp1−/−) MEFs probed with the indicated antibodies. Quantification (bottom) of PKC band intensity normalized to Tubulin loading control.
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Repeated Measures One-Way ANOVA and Tukey HSD Test. IB quantification (excluding (E)) represent the mean ± SEM from at least three independent experiments. Dashed line ((B) and (E)) indicates splicing of irrelevant lanes from a single blot.