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Cell Growth & Differentiation Vol. 12, 465-470, September 2001
© 2001 American Association for Cancer Research

Targeting of Protein Kinase C {delta} to Mitochondria in the Oxidative Stress Response1

Pradip K. Majumder, Neerad C. Mishra, Xiangao Sun, Ajit Bharti, Surender Kharbanda, Satya Saxena and Donald Kufe2

Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [P. K. M., X. S., A. B., S. K., D. K.] and Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87115 [N. C. M., S. S.]


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 References
 
The cellular response to oxidative stress includes the release of mitochondrial cytochrome c and the induction of apoptosis. Here we show that treatment of diverse cells with hydrogen peroxide (H2O2) induces the targeting of protein kinase C {delta} (PKC{delta}) to mitochondria. The results demonstrate that H2O2-induced activation of PKC{delta} is necessary for translocation of PKC{delta} from the cytoplasm to the mitochondria. The results also show that mitochondrial targeting of PKC{delta} is associated with the loss of mitochondrial transmembrane potential and release of cytochrome c. The functional importance of this event is also supported by the demonstration that H2O2-induced apoptosis is blocked by the inhibition of PKC{delta} activation and translocation to mitochondria. These findings indicate that mitochondrial targeting of PKC{delta} is required, at least in part, for the apoptotic response of cells to oxidative stress.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 References
 
Normal cellular metabolism results in the generation of ROS,3 which, through damage to DNA and proteins, activate proapoptotic signals (1 , 2) . The mechanisms responsible for ROS-induced apoptosis are unclear. Studies have suggested that ROS-induced apoptosis is dependent on the p53 tumor suppressor (3 , 4) . Other work on the apoptotic response to oxidative stress has supported the involvement of topoisomerase II (5) , the p66shc adaptor protein (4) , the p85 subunit of phosphatidylinositol 3-kinase (3) , and the c-Abl tyrosine kinase (6) .

Certain insights into ROS-induced signaling have been derived from the finding that PKC{delta} is phosphorylated on tyrosine in cells treated with H2O2 (7, 8, 9) . Significantly, tyrosine phosphorylation of PKC{delta} in the response to ROS confers independence from lipid cofactors for catalytic activity (7) . Phosphorylation of PKC{delta} on Tyr-512 and Tyr-523 has been shown to be important for H2O2-induced activation (7) . Other studies have demonstrated that c-Abl interacts with PKC{delta} in the response to H2O2 and that c-Abl phosphorylates PKC{delta} on Tyr-512 but not Tyr-523 (6) . These findings indicate that ROS induce phosphorylation of PKC{delta} by c-Abl and at least one other tyrosine kinase.

Recent work has demonstrated that H2O2 induces the release of mitochondrial cytochrome c and, thereby, apoptosis (10) . In the present studies, we show that treatment of cells with H2O2 is associated with the targeting of PKC{delta} to mitochondria. The results also demonstrate that activation of PKC{delta} is required for mitochondrial localization and for ROS-induced loss of mitochondrial transmembrane potential, cytochrome c release, and apoptosis.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 References
 
To assess the effects of ROS on the subcellular distribution of PKC{delta}, human U-937 cells were treated with H2O2 and harvested at varying intervals. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting with anti-PKC{delta}. The results demonstrate that treatment with 1 mM H2O2 is associated with decreases in cytoplasmic and concomitant increases in mitochondrial PKC{delta} (Fig. 1A)Citation . The finding that treatment with higher concentrations of H2O2 is associated with increased localization of PKC{delta} to mitochondria indicated that this response is dose-dependent (Fig. 1A)Citation . Immunoblot analysis of the fractions with antibodies against cytosolic ß-actin and mitochondrial Hsp60 was performed to assure purity of the preparations (Fig. 1A)Citation . In contrast with the response of PKC{delta} to H2O2 treatment, there was little if any effect of this agent on mitochondrial levels of PKC{zeta} or PKC{gamma} (Fig. 1B)Citation . To confirm involvement of ROS in the targeting of PKC{delta} to mitochondria, cells were treated with NAC, a scavenger of reactive oxygen intermediates and precursor of glutathione (11 , 12) . The results demonstrate that NAC inhibits H2O2-induced localization of PKC{delta} to mitochondria (Fig. 1C)Citation .



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  		Fig. 1. Mitochondrial translocation of PKC{delta} in response to H2O2 treatment. A, U-937 cells were treated with 1 mM H2O2 for the indicated times (left and middle) or with the indicated concentrations of H2O2 for 1 h (right). Cytosolic and mitochondrial lysates were subjected to immunoblotting with anti-PKC{delta}. Lysates were also analyzed for expression of ß-actin, mitochondrial Hsp60, and cytosolic I{kappa}B{alpha} to assess equal loading of the lanes and purity of the subcellular fractions. B, the same mitochondrial lysates used in A were subjected to immunoblotting with anti-PKC{zeta} and anti-PKC{gamma}. C, U-937 cells were pretreated with NAC for 1 h before adding 1 mM H2O2 for the indicated times. Mitochondrial lysates were analyzed by immunoblotting with anti-PKC{delta} and anti-Hsp60.

 
To extend these findings, intracellular localization of PKC{delta} was visualized with a charge-coupled device camera and image analyzer. Fluorescence detection in control cells showed distinct patterns for PKC{delta} (red signal) and a mitochondrial-selective dye (MitoTracker; green signal; Fig. 2Citation ). The finding that H2O2 treatment is associated with a change in fluorescence signals (red and green -> yellow/orange) provided additional support for translocation of PKC{delta} to mitochondria (Fig. 2)Citation . These findings and those obtained by subcellular fractionation demonstrate that PKC{delta} localizes to mitochondria in the response to oxidative stress.



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  		Fig. 2. Detection of mitochondrial PKC{delta} by immunofluorescence microscopy. U-937 cells were treated with 1 mM H2O2 for 1 h. After washing, control (A) and H2O2-treated (B) cells were immobilized on slides, fixed, and incubated with anti-PKC{delta} and then Texas Red-conjugated goat antirabbit IgG. Mitochondria were stained with the mitochondria-selective permeant dye Mitotracker Green FM.

 
To determine whether PKC{delta} activity contributes to mitochondrial targeting of PKC{delta} in response to H2O2, we treated U-937 cells with the selective PKC{delta} inhibitor, rottlerin (13) . The results demonstrate that H2O2-induced localization of PKC{delta} to mitochondria is attenuated by rottlerin (Fig. 3ACitation , left). To assess applicability of the results to diverse cell types, similar studies were performed on NIH3T3 fibroblasts. As found in U-937 cells, treatment with H2O2 was associated with localization of PKC{delta} to mitochondria (Fig. 3ACitation , right). Moreover, the demonstration that rotterlin inhibits H2O2-induced translocation of PKC{delta} to mitochondria supported involvement of the PKC{delta} kinase function (Fig. 3ACitation , right). To confirm that PKC{delta} activation is necessary for mitochondrial translocation, we transfected 293 cells with vectors expressing GFP, GFP-PKC{delta} or a kinase-inactive GFP-PKC{delta} (K378R) mutant (6) . Analysis of the mitochondrial fraction by immunoblotting with anti-GFP demonstrated H2O2-induced targeting of PKC{delta} to mitochondria (Fig. 3B)Citation . By contrast, H2O2 had no apparent effect on mitochondrial localization of GFP-PKC{delta} (K378R; Fig. 3BCitation ). Mitochondrial targeting of GFP-PKC{delta}, but not GFP-PKC{delta}(K-R), was confirmed by fluorescence microscopy (Fig. 3C)Citation . Thus, overlay of GFP-PKC{delta}, but not GFP-vector or GFP-PKC{delta}(K-R), signals with MitoTracker red showed localization to mitochondria (Fig. 3C)Citation . These findings indicate that activation of the PKC{delta} kinase function is necessary for H2O2-induced mitochondrial localization.



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  		Fig. 3. H2O2-induced translocation of PKC{delta} to mitochondria is dependent on PKC{delta} activation. A, U-937 (left) and NIH3T3 (right) cells were treated with rottlerin (Rot) for 30 min before adding 1 mM H2O2 for 1 h. Mitochondrial lysates were subjected to immunoblot analysis with anti-PKC{delta} and anti-Hsp60. B and C, 293T cells were transfected with vectors expressing GFP, GFP-PKC{delta}, or GFP-PKC{delta} (K-R). At 24 h after transfection, cells were treated with H2O2 for the indicated times (B) or for 60 min (C). B, mitochondrial lysates were analyzed by immunoblotting with anti-GFP and anti-Hsp60 (top and middle). Total cell lysate was subjected to immunoblot analysis with anti-GFP to assess expression of the vectors (bottom). C, cells were immobilized on slides, fixed, and incubated with anti-GFP (left). Mitochondria were stained with mitochondria-selective permanent dye MitoTracker RedCMXRos (middle). Right, overlay of the signals.

 
Other studies have demonstrated that PKC{delta} interacts with the c-Abl tyrosine kinase in the cellular response to oxidative stress (6) . To determine whether c-Abl is necessary for H2O2-induced targeting of PKC{delta} to mitochondria, wild type (c-Abl+/+) and c-Abl-/- MEFs were treated with H2O2. H2O2-induced activation of PKC{delta} was similar in both cells (Fig. 4A)Citation . In addition, H2O2-induced tyrosine phosphorylation of PKC{delta} in the cytosolic and mitochondrial fractions was similar in wild-type and c-Abl-/- cells (Fig. 4B)Citation . Moreover, localization of PKC{delta} to mitochondria was found in the H2O2 response of both cell types (Fig. 4C)Citation . These findings indicate that c-Abl is not required for H2O2-induced PKC{delta} activation and localization to mitochondria.



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  		Fig. 4. H2O2-induced mitochondrial targeting of PKC{delta} is independent of the c-Abl kinase. A, Wild-type (c-Abl+/+) and c-Abl-/- MEFs were treated with 1 mM H2O2 for 1 h. Anti-PKC{delta} immunoprecipitates from total cell lysates were incubated with histone H1 and [{gamma}-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. B, Wild-type (c-Abl+/+) and c-Abl-/- MEFs were treated with 1 mM H2O2 for 1 h. Anti-PKC{delta} immunoprecipitates from mitochondrial and cytosolic fractions were analyzed by immunoblotting with anti-P-Tyr. C, Wild-type (c-Abl+/+) and c-Abl-/- MEFs were treated with 1 mM H2O2 for 1 h. Mitochondrial lysates were analyzed by immunoblotting with anti-PKC{delta} and anti-Hsp60.

 
Treatment of COS cells with H2O2 is associated with cytochrome c release (10) . The finding that H2O2 also induces cytochrome c release in U-937 cells indicated that this response is not restricted by cell type (Fig. 5A)Citation . To determine whether PKC{delta} is functional in inducing release of cytochrome c, U-937 cells were pretreated with NAC or rotterlin. The results demonstrate that NAC blocks H2O2-induced release of cytochrome c (Fig. 5B)Citation . Importantly, pretreatment with rotterlin also blocked the release of cytochrome c in response to H2O2 treatment (Fig. 5B)Citation . These results and those obtained for translocation of PKC{delta} to mitochondria support the involvement of PKC{delta} in H2O2-induced release of cytochrome c.



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  		Fig. 5. H2O2-induced cytochrome c release is regulated by PKC{delta}. A, cytosolic lysates from U-937 cells treated with 1 mM H2O2 for the indicated times were analyzed by immunoblotting with anti-cytochrome c and anti-actin. B, U-937 cells were pretreated with NAC or rottlerin for 30 min before adding 1 mM H2O2 for 1 h. Cytosolic lysates were subjected to immunoblotting with anti-cytochrome c and anti-actin.

 
PKC{delta} consists of an NH2-terminal RD and a COOH-terminal catalytic fragment (14) . To further assess the role of PKC{delta} in H2O2-induced apoptosis, we studied MCF-7 cells that stably express the empty neo vector (MCF-7/neo) or the Mr 35,000 RD (MCF-7/PKC{delta}RD) (15) . In contrast to MCF-7/neo cells, translocation of PKC{delta} to mitochondria was attenuated in H2O2-treated MCF-7 cells stably expressing PKC{delta}RD (Fig. 6A)Citation . Other studies have demonstrated that H2O2 treatment is associated with the loss of mitochondrial transmembrane potential ({Delta}{psi}m; 16 ). To determine whether PKC{delta} is functional in inducing both loss of {Delta}{psi}m and cytochrome c release, H2O2-treated MCF-7/neo and MCF-7/PKC{delta}RD cells were incubated with rhodamine 123. Analysis by flow cytometry demonstrated that H2O2-induced loss of {Delta}{psi}m is attenuated in MCF-7/PKC{delta}RD as compared with MCF-7/neo, cells (Fig. 6B)Citation . The release of cytochrome c in response to PKC{delta} was also attenuated in MCF-7/PKC{delta}RD cells (Fig. 6C)Citation . In concert with these results, H2O2 treatment of MCF-7/neo cells was associated with the induction of apoptosis, and this response was attenuated in the MCF-7/PKC{delta}RD cells (Fig. 6D)Citation . These findings demonstrate that targeting of PKC{delta} to mitochondria contributes to H2O2-induced loss of {Delta}{psi}m, cytochrome c release, and apoptosis.



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  		Fig. 6. PKC{delta} regulates H2O2-induced loss of mitochondrial transmembrane potential, cytochrome c release, and apoptosis. MCF-7/neo and MCF-7/PKC{delta}RD cells were treated with 1 mM H2O2 for the indicated times. A, mitochondrial lysates were analyzed by immunoblotting with anti-PKC{delta} and anti-Hsp60. B, cells were stained with Rhodamine 123 for 30 min and analyzed by flow cytometry. C, cytosolic lysates were subjected to immunoblotting with anti-cytochrome c and anti-actin. D, cells were fixed in ethanol, stained with propidium iodide, and analyzed by FACScan. The results are expressed as the percentage (mean ± SE) of apoptosis as determined from three independent experiments, each performed in duplicate.

 
ROS have been implicated in the regulation of both cell growth and apoptosis (17, 18, 19) . Although the signals activated by ROS are for the most part unclear, previous work has demonstrated that PKC{delta} is phosphorylated on tyrosine in the cellular response to H2O2 treatment (7, 8, 9) . Other studies have shown that c-Abl interacts with PKC{delta} and is in part responsible for tyrosine phosphorylation of PKC{delta} in the response to H2O2 (6) . The available findings indicate that PKC{delta} is activated by ROS and that PKC{delta} phosphorylates and activates c-Abl (6 , 10) . In a potential auto-catalytic loop, c-Abl phosphorylates and further activates PKC{delta} (6) . The present studies demonstrate that ROS induce targeting of PKC{delta} to mitochondria, and that this response is dependent on activation of the PKC{delta} kinase function. These findings are in concert with recent reports showing that phorbol ester-induced activation of PKC{delta} is associated with translocation of PKC{delta} to mitochondria (15 , 20) . However, in contrast to the present results obtained with H2O2, phorbol ester-induced activation of PKC{delta} is not dependent on ROS generation or phosphorylation of PKC{delta} on tyrosine (data not shown). The results also demonstrate that ROS-induced targeting of PKC{delta} to mitochondria occurs in c-Abl-/- cells. These findings indicate that, while c-Abl activation is dependent on PKC{delta} (10) , activation and translocation of PKC{delta} to mitochondria in the response to H2O2 is independent of the c-Abl kinase.

Release of cytochrome c from mitochondria triggers the activation of caspases and the induction of apoptosis (21) . Recent work has demonstrated that the response of cells to oxidative stress includes loss of mitochondrial transmembrane potential and release of cytochrome c (10 , 16) . The available findings also indicate that these effects of ROS on mitochondria are mediated in part by a c-Abl-dependent mechanism (10 , 16) . The results of the present studies show that ROS-induced cytochrome c release is also regulated by activation and translocation of PKC{delta} to mitochondria. Taken together with the demonstration that c-Abl functions in the apoptotic response to oxidative stress (10) , these findings indicate that signaling by both PKC{delta} and c-Abl is needed for ROS-induced loss of mitochondrial transmembrane potential and release of cytochrome c. Indeed, although treatment of c-Abl-/- cells with H2O2 is associated with PKC{delta} activation and localization to mitochondria, these cells failed to respond to oxidative stress with release of cytochrome c and the induction of apoptosis (10) . Finally, PKC{delta} is also activated by PDK1-mediated phosphorylation in the cellular response to serum stimulation (22) . Thus, PKC{delta} seems to be functional in both pro- and antiapoptotic pathways, and therefore it could represent a switch that determines cell fate.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
References
 
Cell Culture and Reagents.
Human U-937 myeloid leukemia cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Human MCF-7, MCF-7/neo, and MCF-7/PKC{delta}RD (15) breast cancer cells, 293T cells, and wild-type and c-Abl-/- MEFs (23) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and antibiotics. Cells were treated with 1 mM H2O2 (Sigma Chemical Co.), 10 µM rottlerin (Sigma Chemical Co.), and 30 mM NAC (Calbiochem). Transfections were performed with Superfect (Qiagen).

Isolation of the Cytosolic Fraction.
Cells were suspended in ice-cold 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM phenylmethylsulphonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 250 mM sucrose. The cells were disrupted by Douce homogenization. After centrifugation at 1500 x g for 5 min at 4°C, the supernatants were centrifuged at 105,000 x g for 30 min at 4°C. The resulting supernatant was used as the soluble cytoplasmic fraction.

Isolation of the Mitochondrial Fraction.
Cells were suspended in ice-cold 5 mM HEPES (pH 7.5), 210 mM mannitol, 1 mM EGTA, 70 mM sucrose, and 110 µg/ml digitonin. The cells were disrupted in a glass homogenizer (Pyrex No. 7727-07) and centrifuged at 2,000 x g for 20 min at 4°C. The pellets were resuspended in the same buffer, homogenized again (Pyrex No. 7726), and centrifuged at 2000 x g for 5 min at 4°C. The supernatants (S1) were collected. The pellets were rehomogenized, centrifuged at 2,000 x g for 5 min, and the resultant supernatants (S2) collected. Supernatants S1 and S2 were pooled and centrifuged at 11,000 x g for 10 min. The mitochondrial pellets were resuspended in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM phenylmethylsulphonyl fluoride, 1 mM DTT, 10 µg/ml leupeptin, and 10 µg/ml aprotinin] for 30 min on ice and then centrifuged at 15,000 x g for 20 min. The supernatant was used as the soluble mitochondrial fraction. Protein concentration was determined by the BioRad protein estimation kit.

Immunoblot Analysis.
Soluble proteins were subjected to immunoblot analysis with anti-PKC{delta} (Santa Cruz Biotechnology), anti-ß-actin (Sigma), anti-Hsp60 (Stressgen), anti-I{kappa}B{alpha} (Santa Cruz), anti-PKC{zeta} (Santa Cruz), anti-PKC{gamma} (Santa Cruz), anti-GFP (Clontech), and anti-cytochrome c (24) . The immune complexes were detected with antirabbit or antimouse IgG peroxidase conjugate (Amersham) and visualized by enhanced chemiluminescence (Amersham Pharmacia).

Immunofluorescence Microscopy.
Cells were plated onto poly-D-lysine-coated glass coverslips. After 24 h, the cells were treated with 1 mM H2O2 for 1 h and then fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 min. Cells were washed with PBS, permeabilized with 0.2% Triton X-100 for 10 min, washed again, and incubated for 30 min in complete medium. The coverslips were incubated with 5 µg/ml anti-PKC{delta} for 1 h and then Texas Red-goat antirabbit Ig (heavy and light chains) conjugate (Molecular Probes, Eugene, OR). Mitochondria were stained with 100 nM MitoTracker Green FM for experiments with GFP; mitochondria were stained with 100 nM MitoTracker Red CMXRos (Molecular Probes). Coverslips were mounted onto slides with 0.1 M Tris (pH 7.0) in 50% glycerol. The cells were visualized by digital confocal immunofluorescence, and images were captured with a charge-coupled device camera mounted on a Zeiss Axiioplan 2 microscope. Images were deconvolved using Slidebook software (Intelligent Imaging Innovations, Inc., Denver, CO).

Assessment of PKC{delta} Activity.
PKC{delta} activity was assayed by incubating anti-PKC{delta} immunoprecipitates in PKC kinase buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 20 µM ATP, 2.5 µCi [{gamma}-32P]ATP, and 200 µg/ml histone H1 (7) for 5 min at 30°C. The reaction products were analyzed by SDS-PAGE and autoradiography.

Analysis of Mitochondrial Membrane Potential.
Cells were treated with 1 mM H2O2 and incubated with 50 ng/ml Rhodamine 123 (Molecular Probes) for 30 min at 37°C. After washing with PBS, cells were analyzed by flowcytometry using 488 nm excitation and a 575/26 ethidium bandpass filter.

Assessment of Apoptosis.
Cells were fixed with 80% ethanol, washed, and incubated with 2.5 µg/ml propidium iodide and 50 µg/ml RNase. Cells with sub-G1 DNA were determined by FACScan (Becton Dickinson).


   Acknowledgments
 
The authors appreciate the technical assistance of Kamal Chauhan.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Public Health Service Grant CA42802, awarded by the National Cancer Institute, NIH, and the Department of Health and Human Services and by the Office of Health and Biological Research, U.S. Department of Energy, Cooperative Agreement DE-FCO4-96AL76406. Back

2 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115. Phone: (617) 632-3141; Fax: (617) 632-2934. Back

3 The abbreviations used are: ROS, reactive oxygen species; PKC, protein kinase C; H2O2, hydrogen peroxide; NAC, N-acetyl-L-cysteine; MEF, mouse embryo fibroblast; GFP, green fluorescence protein; RD, regulatory domain. Back

Received for publication 2/ 8/01. Revision received 6/21/01. Accepted for publication 6/21/01.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 

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