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J. Biol. Chem., Vol. 275, Issue 29, 21793-21796, July 21, 2000

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ACCELERATED PUBLICATION
Mitochondrial Translocation of Protein Kinase C  in Phorbol Ester-induced Cytochrome c Release and Apoptosis^*

Pradip K. Majumder, Pramod Pandey, Xiangao Sun, Keding Cheng, Rakesh Datta, Satya Saxena, Surender Kharbanda, and Donald Kufe^§

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and the  Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87115

Received for publication, January 20, 2000, and in revised form, May 12, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC)  from the cytoplasm to mitochondria. The results also show that translocation of PKC results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKC translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKC to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The protein kinase C (PKC)¹ family of serine/threonine protein kinases is involved in intracellular signals that regulate growth, differentiation, and apoptosis. The PKC isoforms have been subdivided into: (i) the conventional PKCs (cPKCs; , , ), which are dependent on calcium and activated by diacylglycerol (DAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA); (ii) the novel PKCs (nPKCs; , , , ), which are calcium-independent and activated by DAG or TPA; and (iii) the atypical PKCs (aPKCs; , ) which are calcium-independent and not activated by DAG or TPA (1). The cPKCs are cleaved in the third variable region by calpains I and II to catalytically active fragments (2). Other studies (2) have demonstrated that the nPKC, PKC, is cleaved in the third variable region by caspase 3 in the apoptotic response of cells to DNA damage and engagement of the tumor necrosis factor receptor. The cleaved catalytic fragment of PKC is constitutively activated and, when overexpressed in HeLa and other cells, is sufficient to induce apoptosis (3). Proteolytic activation of PKC has also been implicated in ultraviolet radiation-induced apoptosis of keratinocytes (4). These findings have supported a role for the PKC isoform in the apoptotic response of cells to diverse stimuli.

The treatment of human myeloid leukemia cells with TPA is associated with the induction of apoptosis (5). Other studies have demonstrated that TPA induces apoptosis of human MCF-7 breast cancer cells and Jurkat T cells (6, 7). These findings have suggested that TPA-induced activation of certain PKC isoforms confers signals that induce apoptosis. However, the identity of the PKC isoform(s) that are activated in TPA-induced apoptosis and the downstream signals that confer the apoptotic response are unknown. The available evidence indicates that at least two pathways induce apoptosis in the cellular response to other stimuli. One pathway is initiated by engagement of the tumor necrosis factor or Fas receptors and thereby activation of caspase 8 (8, 9). In turn, caspase 8 cleaves Bid and induces cytochrome c release (10, 11). Caspase 8 can also directly activate caspase 3 (12). In the second pathway, other signals, which importantly remain undefined, converge to induce the release of cytochrome c (13, 14). Cytosolic cytochrome c binds to Apaf-1, induces the autoprocessing of caspase 9 and thereby the activation of caspase 3 (11, 15). Neither of these pathways has been linked to TPA-induced apoptosis.

The present studies demonstrate that TPA treatment is associated with the translocation of cytoplasmic PKC to mitochondria. The results show that translocation of PKC induces the release of cytochrome c and the activation of caspase 3. These findings support a novel mechanism for TPA-induced cytochrome c release and apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cell Culture and Reagents-- Human U-937 myeloid leukemia cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. MCF-7, MCF-7/neo, MCF-7/PKCRD, and 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells (3 × 10⁶/150-mm culture dish) were plated 24 h before treating with 250 nM TPA (Sigma), 100 nM bryostatin 1 (ICN, Aurora, OH), 10 µM 1,2-dioctanoyl-sn-glycerol (DOG; Calbiochem) and 0.5 unit/ml phospholipase C (PLC; Sigma). Cells were also treated with 10 µM rottlerin (Calbiochem).

Isolation of Mitochondria-- Cells were washed twice with phosphate-buffered saline (PBS), homogenized in buffer A (210 mM manitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA) and 110 µg/µl digitonin in a glass homogenizer (Pyrex no. 7727-07) and centrifuged at 5000 × g for 20 min. Pellets were resuspended in buffer A, homogenized in a small glass homogenizer (Pyrex no. 7726), and centrifuged at 2000 × g for 5 min. Supernatant (S1) was collected and the pellet again homogenized in buffer A. Supernatant (S2) was collected after centrifugation at 2000 × g for 5 min. Supernatants S1 and S2 were mixed and centrifuged at 11,000 × g for 10 min. Mitochondrial pellets were disrupted in lysis buffer at 4 °C for 30 min and then centrifuged at 15,000 × g for 20 min. The concentration of mitochondrial proteins in the supernatant was determined using Bio-Rad protein estimation kit.

Isolation of the Cytosolic Fraction-- Cells were washed twice with PBS, and the pellet was suspended in 5 ml of ice-cold buffer B (20 mM HEPES, pH 7.5, 1.5 mM MgCl₂, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin and aprotinin) containing 250 mM sucrose. The cells were homogenized by disrupting three times in a Dounce homogenizer in buffer B. After centrifugation for 5 min at 4 °C, the supernatants then were centrifuged at 105,000 × g for 30 min at 4 °C. The resulting supernatant was used as the soluble cytosolic fraction.

Immunoprecipitation and Immunoblot Analysis-- Total, cytoplasmic or mitochondrial lysates were subjected to immunoprecipitation with anti-GFP, anti-PKC (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PKCµ (Santa Cruz), anti-PKC (Upstate Biotechnology, Inc.), anti-PKC (Santa Cruz), anti-PKC (Santa Cruz), anti-PKC (Santa Cruz), or anti-PKC (Santa Cruz) antibodies. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The residual binding sites were blocked by incubating the filters with 5% nonfat dry milk in PBST (PBS, 0.05% Tween 20). The filters were incubated with anti-PKC, anti-cytochrome c (16), anti-Hsp-60 (Stressgen, Victoria, British Columbia, Canada), anti-actin (Sigma), anti-PKC, anti-PKCµ, anti-PKC, or anti-GFP (CLONTECH, Palo Alto, CA). After washing twice with PBST, the filters were incubated with anti-rabbit or anti-mouse IgG peroxidase conjugate and developed by ECL (Amersham Pharmacia Biotech).

Plasmids-- pEGFP-PKC and PKC-RD were prepared as described previously (17). The pEGFP-PKC(K378R) mutant was generated by site-directed mutagenesis.

Transient Transfections-- 293T cells were transiently transfected with empty vector (pEGFP-C1), pEGFP-PKC, or pEGFP-PKC (K378R) using SuperFect (Qiagen). At 24 h after transfection, cells were lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 µg/ml leupeptin and aprotinin) and subjected to immunoblotting with anti-PKC and anti-GFP. Signal intensities were determined by densitometric analysis.

Immunofluorescence Microscopy-- Cells immobilized on slides were fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, incubated with 20 ng of anti-PKC/slide and then Texas Red-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates, Inc). Mitochondria were stained with 0.006 ng/slide of Mitotracker Green FM (Molecular Probes). The slides were analyzed using a Zeiss Auxiphot fluorescence microscope coupled to a CCD camera and a power Macintosh 8100. Image analysis was performed using the IPLab Spectrum 3.1 software (Signal Analytics).

PKC Activity Assays-- 293T cells were transiently transfected with GFP-PKC or GFP-PKC(K-R). Total cell lysates were subjected to immunoprecipitation with anti-PKC, anti-PKC, anti-PKC, anti-PKC, anti-PKCµ, or anti-PKC. The immune complex kinase assays were performed using H1 histone as a substrate as described previously (18).

Quantitation of Apoptosis by Flow Cytometric Analysis-- Cells were harvested, washed twice with PBS, and fixed with 80% ethanol. Cells (10⁶ cells/ml) were washed and incubated with propidium iodide (2.5 µg/ml) and RNase (50 µg/ml). FACScan (Becton Dickinson) was used to assess cells with sub-G₁ DNA content.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

PKC isoforms regulate diverse cellular processes but are not known as effectors of mitochondria (1). To determine whether PKC regulates mitochondrial function, human U-937 cells were treated with TPA to activate PKC. PKC translocation was assessed by subjecting cytoplasmic and mitochondrial fractions to immunoblotting with anti-PKC antibodies. The results demonstrate that TPA treatment is associated with decreases in cytoplasmic PKC and concomitant increases in mitochondrial PKC (Fig. 1A). As controls, the cytoplasmic and mitochondrial fractions were also subjected to immunoblotting with anti-actin and anti-Hsp60 to ensure purity of the preparations (Fig. 1A). By contrast to translocation of PKC, TPA had no detectable effect on cytoplasmic or mitochondrial levels of PKC and PKC (Fig. 1B and data not shown). The immunoblots were scanned to calculate percent PKC translocation to mitochondria. The results demonstrate that approximately 40% of PKC translocates to mitochondria in response to TPA.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Translocation of PKC to mitochondria in response to TPA treatment. A, U-937 cells were treated with 250 nM TPA for the indicated times. The cells were harvested and separated into cytosolic (Cyto) and mitochondrial (Mito) fractions. Proteins were subjected to 10% SDS-PAGE and immunoblot analysis with anti-PKC. As controls, lysates were also analyzed by immunoblotting with anti-Actin or anti-Hsp60. B, U-937 cells were treated with 250 nM TPA for the indicated times. Cytosolic (Cyto) and mitochondrial (Mito) fractions were analyzed by immunoblotting with anti-PKC or anti-PKC.

The demonstration that PKC also translocates to mitochondria in TPA-treated MCF-7 cells indicates that the finding is not restricted to certain cell types (Fig. 2A). In addition, to confirm the subcellular redistribution of PKC in TPA-treated cells, we visualized intracellular fluorescence with a CCD camera and image analyzer. Examination of fluorescence markers in control cells showed distinct patterns for anti-PKC (red signal) and a mitochondrion-selective dye (Mitotracker; green signal) (Fig. 2B). The demonstration that TPA induces a marked change in fluorescence signals (red and green  yellow/orange) supported translocation of PKC to mitochondria (Fig. 2B). These findings obtained by immunofluorescence microscopy thus confirm the results of PKC redistribution found by subcellular fractionation.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Translocation of PKC to mitochondria in response to TPA treatment. A, MCF-7 cells were treated with 250 nM TPA for 1 h. Cytosolic (Cyto) and mitochondrial (Mito) fractions were analyzed by immunoblotting with anti-PKC. B, U-937 cells were treated with TPA for 1 h. After washing, the cells were immobilized on slides, fixed, and incubated with anti-PKC followed by Texas Red-conjugated goat anti-rabbit IgG. Mitochondria were stained with the mitochondria-selective permeant dye Mitotracker Green FM.

To determine whether the natural product bryostatin, which activates PKC (19), also induces the translocation of PKC, mitochondrial lysates from U-937 cells treated with 100 nM bryostatin were subjected to immunoblot analysis with anti-PKC. As a control, mitochondrial lysates were also subjected to immunoblot analysis with anti-PKC. The results demonstrate that, in contrast to PKC, treatment with bryostatin was associated with translocation of PKC to mitochondria (Fig. 3A). PLC is activated by cell membrane-initiated signaling pathways and, by conferring the hydrolysis of phosphatidylinositol or phosphatidylcholine, results in the formation of DAG (20). To determine whether PLC induces the translocation of PKC, mitochondrial lysates from U-937 cells treated with 0.5 unit/ml PLC were subjected to immunoblot analysis with anti-PKC. The results demonstrate that treatment with PLC is associated with translocation of PKC to mitochondria (Fig. 3B). To confirm the involvement of DAG in mitochondrial translocation of PKC, cells were treated with a cell permeable-DAG (DOG) (21). Immunoblot analysis of DOG-treated cell lysates demonstrated that DOG induced the translocation of PKC to mitochondria (Fig. 3C). These findings indicate that, like TPA, treatment with bryostatin, PLC, and DOG is associated with redistribution of cytosolic PKC to mitochondria.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Translocation of PKC to mitochondria in response to bryostatin, PLC, or DOG treatment. A, U-937 cells were treated with bryostatin (Bryo) for the indicated times. Proteins from the mitochondrial (Mito) fraction were subjected to immunoblot analysis with anti-PKC or anti-PKC. As control, mitochondrial lysates were also analyzed by immunoblotting with anti-Hsp60. B and C, U-937 cells were treated with 0.5 unit/ml PLC (B) or 10 µM DOG (C) and harvested at the indicated times. Proteins from the mitochondrial fraction were subjected to immunoblot analysis with anti-PKC. As control, mitochondrial lysates were also analyzed by immunoblotting with anti-Hsp60.

To determine whether activation of PKC is required for translocation to mitochondria, we transfected cells with a vector expressing GFP-tagged PKC. Immunoblot analysis with anti-GFP demonstrated no detectable PKC in the mitochondrial fraction from cells transfected with an empty GFP vector (Fig. 4A). By contrast, transfection of kinase-active GFP-PKC was associated with PKC expression in mitochondria (Fig. 4A). Moreover, treatment of the GFP-PKC-transfected cells with TPA resulted in further increases in levels of mitochondrial PKC (Fig. 4A). Significantly, transfection of kinase-inactive GFP-PKC(K-R) had no effect on expression of mitochondrial PKC (Fig. 4A). In addition, overexpression of GFP-PKC(K-R) blocked the TPA-induced translocation of PKC to mitochondria (Fig. 4A). To demonstrate that PKC(K-R) specifically blocks endogenous PKC activity, and not that of other isoforms of PKC, 293T cells were transiently transfected with GFP-PKC or GFP-PKC(K-R). Following transfection, cell lysates were subjected to immunoprecipitation with anti-PKC, anti-PKCµ, anti-PKC, anti-PKC, anti-PKC, or anti-PKC. The precipitates were assayed in in vitro kinase assays using H1 histone as substrate. The results demonstrate that, in contrast to PKCµ, PKC, PKC, or PKC, overexpression of PKC(K-R) specifically inhibits the activity of endogenous PKC (Fig. 4B). Of note, the results also indicate that overexpression of PKC(K-R) is associated with slight inhibition of the phosphorylated and active PKC (Fig. 4B). PKC consists of an N-terminal regulatory domain (RD) and a C-terminal catalytically active fragment (2). MCF-7 cells stably transfected to express the 35-kDa RD exhibit attenuation of TPA-induced PKC activity.² Translocation of PKC to mitochondria was also attenuated in TPA-treated MCF-7/PKCRD cells as compared with that in MCF-7 cells expressing the empty neo vector (Fig. 5A). Other studies were performed with rottlerin, a selective inhibitor of PKC activation (22). Treatment of U-937 cells with rottlerin abrogated TPA-induced localization of PKC to mitochondria (Fig. 5B). These findings collectively demonstrate that PKC activation is necessary for its translocation to mitochondria.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   PKC kinase function is necessary for TPA-induced translocation to mitochondria. A, 293T cells were transiently transfected to express empty vector, PKC or PKC(K-R). After 24 h, the cells were treated with TPA for 1 h. Lysates prepared from mitochondria (upper panel) or intact cells (lower panel) were subjected to 10% SDS-PAGE and immunoblot analysis with anti-GFP. B, 293T cells were transfected with GFP-PKC (wt) or GFP-PKC (K-R). Following transfection, total cell lysates were subjected to immunoprecipitation with anti-PKC, anti-PKCµ, anti-PKC, anti-PKC, anti-PKC, or anti-PKC. The precipitates were assayed for phosphorylation of H1 histone (upper panels). Anti-PKC, anti-PKCµ, anti-PKC, anti-PKC, anti-PKC, or anti-PKC immunoprecipitates were analyzed by immunoblotting with anti-PKC, anti-PKCµ, anti-PKC, anti-PKC, anti-PKC, or anti-PKC, respectively (middle panels). Total Lysates were also analyzed by immunoblotting with anti-GFP (lower panels).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   A, MCF-7/neo and MCF-7/PKCRD cells were treated with TPA for the indicated times. Mitochondrial lysates were subjected to immunoblot analysis with anti-PKC. B, U-937 cells were treated with TPA for 1 h or 10 µM rottlerin (ROT) for 1.5 h. Cells were also treated with rottlerin for 0.5 h before adding TPA for an additional 1 h (ROT/TPA). Mitochondrial lysates were subjected to immunoblot analysis with anti-PKC.

The potential role of PKC translocation was explored by assessing mitochondrial release of cytochrome c. Whereas diverse apoptotic signals induce cytochrome c release, phorbol ester treatment of cells has not been associated with this event. Immunoblot analysis of cytoplasmic fractions with anti-cytochrome c demonstrated that TPA treatment of U-937 cells is associated with cytochrome c release (Fig. 6A). Similar results were obtained when U-937 cells were treated with PLC or DOG (Fig. 6, B and C). To determine whether PKC functions in inducing cytochrome c release, we pretreated U-937 cells with rottlerin before adding TPA. Of note, treatment of cells with rottlerin alone is associated with cytotoxic effects that contribute to a detectable release of cytochrome c (Fig. 7A). By contrast, analysis of cytoplasmic lysates demonstrated that rottlerin significantly blocks TPA-induced cytochrome c release (Fig. 7A). As these findings indicate that the PKC kinase function is required for TPA-induced release of cytochrome c, 293T cells were transfected to express GFP, GFP-PKC, or GFP-PKC(K-R) and then treated with TPA. Immunoblotting of the cytoplasmic fraction from GFP positive cells demonstrated abrogation of TPA-induced cytochrome c release in cells expressing PKC(K-R) compared with that in cells transfected with the GFP-PKC vector (Fig. 7B). Taken together, these results and those obtained for PKC translocation support a role for PKC in the mitochondrial release of cytochrome c.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Regulation of TPA-induced cytochrome c release by PKC. A, U-937 cells were treated with TPA for the indicated times. Cytosolic lysates were subjected to immunoblot analysis with anti-cytochrome c (cyt c). B and C, U-937 cells were treated with 0.5 unit/ml PLC (B) or 10 µM DOG (C) for the indicated times. Cytosolic lysates were subjected to immunoblot analysis with anti-cytochrome c. As control, cytoplasmic lysates were also analyzed by immunoblotting with anti-actin.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   A, U-937 cells were treated with TPA for 6 h or rottlerin (ROT) for 6.5 h. Cells were also treated with rottlerin for 0.5 h before adding TPA for an additional 6 h (ROT/TPA). Total cell lysates were analyzed by immunoblotting with anti-cytochrome c (upper panel). Signal intensities from the anti-cytochrome c immunoblotting experiments described in the upper panel were analyzed by densitometric scanning. The signal intensities are expressed in arbitrary value as the mean ± S.D. of three independent experiments (lower panel). B, 293T cells were transiently transfected to express empty vector, PKC or PKC(K-R). After 24 h, the cells were treated with TPA for 3 h. Cytosolic lysates were analyzed by immunoblotting with anti-cytochrome c.

The release of cytochrome c from mitochondria triggers activation of caspases and induction of apoptosis (23). To determine whether TPA-induced PKC translocation and thereby cytochrome c release contributes to apoptosis, U-937 cells treated with rottlerin and TPA were assayed for sub-G₁ DNA content. The results demonstrate that treatment with rottlerin alone induces a low level of apoptosis (Fig. 8A). By contrast, the apoptotic response of U-937 cells to TPA was significantly attenuated by inhibition of PKC with rottlerin (Fig. 8A). Moreover, treatment of MCF-7/neo cells with TPA was also associated with the induction of apoptosis (Fig. 8B). By contrast, the apoptotic response to TPA was significantly attenuated in MCF-7/PKCRD cells (Fig. 8B). Taken together with our other findings, these results support a role for TPA-induced localization of PKC to mitochondria and in the induction of apoptosis.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Regulation of TPA-induced apoptosis by PKC. A, U-937 cells were treated with TPA for 12 h or rottlerin (ROT) for 12.5 h. Other cells were treated with rottlerin for 0.5 h before adding TPA for an additional 12 h (ROT/TPA). Cells were fixed in 80% ethanol, stained with propidium iodide, and analyzed by FACScan. B, MCF-7/neo and MCF-7/PKCRD cells were treated with TPA for 12 h. Untreated (solid bars) and TPA-treated (hatched bars) cells were fixed in ethanol, stained with propidium iodide, and analyzed by FACScan. The results are expressed as mean ± S.D. percentage apoptosis determined from two independent experiments each performed in duplicate.

Previous work has demonstrated that TPA treatment is associated with translocation of PKC to the cell membrane (24). The present studies demonstrate that TPA treatment of diverse cell types is associated with translocation of PKC to mitochondria. These findings have been confirmed by cell fractionation and immunofluorescence studies. The results further demonstrate that the PKC kinase function is necessary for TPA-induced mitochondrial localization. The functional significance of PKC translocation to mitochondria is supported by the finding that this event is linked to mitochondrial release of cytochrome c. Moreover, the results demonstrate that abrogation of PKC translocation to mitochondria significantly inhibits TPA-induced apoptosis. These findings thus support a model in which TPA induces the release of cytochrome c and thereby apoptosis by a PKC-dependent mechanism.

	FOOTNOTES

^* This work was supported by Public Health Service Grants CA42802 (to D. K.) and GM58200 (to R. D.) awarded by the NCI, DHHS and by Charlotte Geyer Foundation Grant 9219401 (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: donald_kufe@dfci.harvard.edu.

Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.C000048200

² P. K. Majumder, P. Pandey, X. Sun, K. Cheng, R. Datta, S. Saxena, S. Kharbanda, and D. Kufe, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; TPA, 12-0-tetradecanoylphorbol-13-acetate; DAG, diacylglycerol; PLC, phospholipase C; DOG, 1,2-dioctanoyl-sn-glycerol; PBS, phosphate-buffered saline; GFP, green fluorescence protein; PAGE, polyacrylamide gel electrophoresis; RD, regulatory domain.

	REFERENCES

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				K.-i. Tanaka, W. Tomisato, T. Hoshino, T. Ishihara, T. Namba, M. Aburaya, T. Katsu, K. Suzuki, S. Tsutsumi, and T. Mizushima Involvement of Intracellular Ca2+ Levels in Nonsteroidal Anti-inflammatory Drug-induced Apoptosis J. Biol. Chem., September 2, 2005; 280(35): 31059 - 31067. [Abstract] [Full Text] [PDF]

				E. N. Churchill, C. L. Murriel, C.-H. Chen, D. Mochly-Rosen, and L. I. Szweda Reperfusion-Induced Translocation of {delta}PKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation Circ. Res., July 8, 2005; 97(1): 78 - 85. [Abstract] [Full Text] [PDF]

S. M. L. Tse, D. Mason, R. J. Botelho, B. Chiu, M. Reyland, K. Hanada, R. D. Inman, and S. Grinstein Accumulation of Diacylglycerol in the Chlamydia Inclusion Vacuole: POSSIBLE ROLE IN THE INHIBITION OF HOST CELL APOPTOSIS J. Biol. Chem., July 1, 2005; 280(26): 25210 - 25215. [Abstract] [Full Text]