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First published online 19 February 2008
doi: 10.1242/jcs.024653

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The PKC -Abl complex communicates ER stress to the mitochondria – an essential step in subsequent apoptosis

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. PKC translocates to the ER following ER stress. (A) Neuro2a cells were treated with Tm (5 µg/ml). Cell lysates from the ER and mitochondria-enriched fractions were subjected to western blot analysis at the indicated times. (Top panel) Changes in the level of PKC in the ER and the mitochondrial fractions at the indicated time points. *P<0.05 vs control in ER fractions, #P<0.05 vs control in mitochondrial fractions (n=3). (Centre panel) Representative western blot of PKC in the two fractions. Calnexin and TOM20 (markers of ER and mitochondria, respectively) were used as internal loading controls for quantification. (Bottom panel) The relative purity of the cellular fractionations was determined by the presence of calnexin, TOM20 and enolase (cytosolic marker). (B) Neuro2a cells were treated with Tm (5 µg/ml) or Tg (3 µM). (Top panel) Representative confocal microscopy image of PKC (red) and PDI (green), demonstrating increased colocalization (yellow) of PKC in the ER following 3 minutes of Tm or Tg treatment. Original magnification was x60. The data are from three independent experiments. (Bottom panel) Representative confocal microscopy image of PKC (red) and mitotracker (green) after 3 minutes or 3 hours of Tm treatment. The data are from two independent experiments. (C) Rats were subjected to 2 hours MCAO followed by 10-240 minutes of reperfusion (I/R). Lysates from the penumbra area of the ipsilateral hemisphere of the rats brains were fractionated. (Top panel) ER fractions were subjected to western blotting using PKC antibody. Calnexin was used as an internal loading control for quantification. (Centre panel) Histogram depicting the amount of PKC associated with the ER in brain samples (PKC/calnexin). Data are expressed as the mean ± s.e. from results of four rats, *P<0.05; **P<0.01, vs sham-operated rats. (Bottom panel) Confirmation of purity of fractionations by using the specific protein markers as described in Fig. 1A. (D) ER localization of PKC determined by immunoelectron microscopy. Representative electron microscopy image of PKC staining in the ER fractions from brains subjected to 2 hours MCAO followed by 4 hours of reperfusion (I/R). (magnification x35,000). Samples were probed in the presence (+) or absence (–) of PKC antibody. Arrows indicate PKC-positive staining with gold particles. Quantification of gold particles associated with ER lumen are provided in the lower histogram. Five random fields of each section from three animals were counted. Data represent the mean ± s.e. from results of three animals per group, *P<0.05 vs sham-operated rats.

View larger version (31K): [in this window] [in a new window]   Fig. 2. V1-1 inhibits ER-stress-mediated apoptosis. Rats were subjected to 2 hours MCAO followed by 24 hours of reperfusion (I2/R24). V1-1 and TAT control (0.2 mg/kg) were injected intraperitoneally at the onset of reperfusion. Neuro2a cells were pre-treated with V1-1 or TAT control (1 µM) for 15 minutes followed by Tm (5 µg/ml) or Tg (3 µM) treatment. (A) (Left panel) ER fractions were isolated from brain. (Right panel) After 3 minutes of Tm treatment, ER fractions were isolated from Neuro2a cells. The lysates were subjected to western blot analysis using anti-PKC antibody. Calnexin was used as internal loading control. (B) Induction of JNK phosphorylation was detected in total-cell lysates of rat brains. (C) Phosphorylation of JNK in total lysates of Neuro2a cells was detected by western blot after 24 hours of Tm (left) or 18 hours of Tg (right) treatment. (D) Phosphorylation of Jun, a downstream effector of JNK, was detected by western blotting at the indicated time points after Tm (upper) or Tg (lower) treatment. (E) TUNEL assay was carried out after 30 hours of Tm or Tg treatment. (Left panel) Representative data of three independent experiments. Original magnification was x40. (Right panel) TUNEL-positive cells are expressed as a percentage of the number of total cells, as determined by staining with Hoechst dye. In the animal study, data are expressed as mean ± s.e. from results of six rats per group. *P<0.05 vs TAT treatment; #P<0.05 vs sham-operated rats. In the cell culture study, the data are presented as mean ± s.e. of three independent experiments. #P<0.05 vs control, *P<0.05 vs Tm treatment, **P<0.01 vs TAT treatment.

View larger version (49K): [in this window] [in a new window]   Fig. 3. ER stress induces the interaction of PKC and Abl. Neuro2a cells were pretreated with V1-1 or TAT control (1 µM) for 15 minutes followed by Tm (5 µg/ml) treatment at the indicated time points. ER and mitochondrial (mito) fractions were then isolated. (A) The levels of Abl in the ER and mitochondria were detected by western blot. (Top) Graph depicting the levels of Abl in the ER and the mitochondrial fractions at the indicated time points. *P<0.05 vs control in ER fractions; #P<0.05 vs control in mitochondria fractions. (Bottom) Representative western blot of staining for Abl in ER and mitochondrial fractions of two independent experiments. Calnexin (ER marker) and TOM20 (mitochondrial marker) were used as loading controls. (B) (Top) ER and mitochondrial fractions were subjected to immunoprecipitation (IP) with anti-Abl antibody and the immunoprecipitates were analyzed by immunoblotting (IB) with anti-PKC and anti-Abl antibodies at the indicated time points. Shown are representative data of four independent experiments. (Bottom) Immunoprecipitates obtained using anti-PKC antibody were analyzed by immunoblotting (IB) using anti-Abl and anti-PKC antibodies at the indicated time points. Shown are representative data of two independent experiments. (C) ER fractions were isolated from the penumbra area of rats brains that had been subjected to 2 hours MCAO followed by 24 hours of reperfusion. Immunoprecipitates obtained using anti-Abl antibody were analyzed by immunoblotting with anti-PKC and anti-Abl antibodies. Data are representative of results obtained from five rats per group.

View larger version (34K): [in this window] [in a new window]   Fig. 4. PKC and Abl are both required for the response to ER stress. Neuro2a cells were transfected with control siRNA, Abl siRNA or PKC siRNA. After 48 hours, cells were pretreated with V1-1 (1 µM) for 15 minutes followed by Tm (5 µg/ml) treatment. (A) Total-cell lysates were analyzed by western blotting to confirm knockdown of Abl (upper panel) and PKC (lower panel). GAPDH in total lysates was used as internal loading control. (B) (Top two panels) Levels of PKC were analyzed by western blotting in the ER (upper panel) and mitochondrial (middle panel) fractions at the indicated time. (Lower panel) Histogram depicting the amount of PKC associated with the ER or mitochondria in Neuro2a cells. Data are expressed as the mean ± s.e. of three independent experiments. *P<0.05 vs Tm treatment, #P<0.05 vs Tm treatment in cells transfected with control siRNA. (C) Representative confocal images of PKC (red) and PDI (green) following 3 minutes of Tm treatment in the cells transfected with control or Abl siRNA. The data are from three independent experiments. Original magnification was x60. (D) Levels of Abl in mitochondrial fractions were analyzed by western blot after 3 hours of Tm treatment in cells transfected with control siRNA or PKC siRNA. Shown are representative data of three independent experiments. (E) Cell lysates from the ER fractions were subjected to immunoprecipitation (IP) with anti-PKC. In vitro kinase assays were carried out with or without V1-1 or knockdown of Abl using histone as a substrate. Data are representative of two independent experiments. (F) Cell lysates from the ER fractions were subjected to immunoprecipitation (IP) using anti-PKC antibody. The immunoprecipitates were analyzed by immunoblotting (IB) using anti-P-Tyr and anti-PKC antibodies. Data are representative for three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5. PKC and Abl mediate ER-stress-induced mitochondria-dependent apoptosis. Neuro2a cells were transfected with control siRNA, or siRNA targeting Abl or PKC. After 48 hours, cells were pretreated with V1-1 (15 minutes, 1 µM) followed by treatment with Tm (5 µg/ml). (A) After 24 hours of Tm treatment, total cell lysates were subjected to western blotting with the indicated antibodies. Data are representative results of three independent experiments. (B) Mitochondrial fractions were isolated from Neuro2a cells treated with Tm for 24 hours after transfection, and subjected to western blot with anti-Bax antibody. TOM20 was used as loading control. Shown are from three independent experiments. (C) TUNEL assay. Neuro2a cells were treated with Tm for 30 hours before staining with TUNEL. Representative data are from two independent experiments. Original magnification was x40.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Disassociation of the PKC-Abl complex by rottlerin reduces ER-stress-induced mitochondrial-dependent apoptosis. Neuro2a cells were treated with Tm (5 µg/ml). After 30 minutes, cells were treated with rottlerin (5 µM). (A) ER and mitochondrial fractions were isolated after a total of 3 hours of Tm treatment. Immunoprecipitates obtained by using anti-Abl antibodies were analyzed by immunoblotting using anti-PKCantibody. Representative data are from three independent experiments. (B) After 24 hours of Tm treatment, mitochondrial and cytosolic fractions were subjected to western blotting using anti-Bax and anti-cytochrome c antibodies, respectively. Tom20 and GAPDH antibodies were used as loading controls for each fraction. Data are representative for two independent experiments. (C) Neuro2a cells were treated with Tm for 30 hours before staining with TUNEL. (Top) Representative data of three independent experiments. Original magnification was x40. (Bottom) TUNEL-positive cells are expressed as a percentage of the number of total cells, as determined by staining after fixation with Hoechst dye. *P<0.05 vs Tm treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Steps in ER-stress-induced apoptosis and communication of this signaling event from the ER to the mitochondria by the PKC-Abl complex. Several steps in the transduction of ER stress signaling leading to apoptosis have been identified. (Step 1) ER stress triggers translocation of PKC to the ER where it binds to Abl and is (directly or indirectly) phosphorylatedon tyrosine residues by Abl. (Step 2) The PKC-Abl complex translocates to mitochondria in a process that depends on PKC catalytic activity. Abl is required for PKC translocation to the mitochondria and, vice versa, PKC is required for Abl translocation to this organelle. (Step 3) PKC-Abl complex activates JNK-mediated mitochondrium-dependent apoptotic cascade. The PKC-specific peptide inhibitor V1-1, inhibits PKC translocation to the ER and the subsequent interaction of PKC and Abl, thereby suppressing ER-stress-mediated apoptosis. Rottlerin, an inhibitor of PKC catalytic activity, blocks the translocation of PKC-Abl complex to mitochondria and, thus, inhibits ER-stress-induced mitochondrium-dependent apoptosis.