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[Cancer Research 63, 1153-1156, March 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Phospholipid Scramblase 3 Is the Mitochondrial Target of Protein Kinase C {delta}-induced Apoptosis1

Jihua Liu, Jun Chen, Qiang Dai and Ray M. Lee2

Huntsman Cancer Institute and Section of Oncology, Department of Medicine, University of Utah, Salt Lake City, Utah 84112


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Protein kinase C (PKC) {delta} translocates to mitochondria during apo-ptosis,but its mitochondrial target remains unclear. We found that PKC-{delta} physically interacted with and phosphorylated phospholipid scramblase 3 (PLS3) after UV irradiation. PLS3 is a high-affinity substrate for PKC-{delta} in vitro with the Km at 10.5 nM. Cells expressing wild-type PLS3 became apoptotic on phorbol ester stimulation, whereas the control cells did not. Expression of a mitochondrial-targeted PKC-{delta} enhanced apoptosis more prominently in HeLa-PLS3 cells than control HeLa cells and HeLa cells expressing an inactive PLS3 mutant. These results indicate that PLS3 is a downstream effector of PKC-{delta} in the mitochondria.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Members of the PKC3 family of kinases play diverse roles in cell signaling, proliferation, apoptosis, and other cellular processes (1 , 2) . PKC-{delta} is particularly associated with apoptosis (3, 4, 5, 6, 7, 8) . PKC-{delta} is present in the cytoplasm, and translocates to various organelles, including the nucleus, mitochondria, and the plasma membrane, on apoptotic stimulation (3 , 5 , 6) . One substrate of PKC-{delta} is PLS1 in the plasma membrane (9) . However, the consequence of PLS1 phosphorylation and how PLS1 contributes to apoptosis remain elusive. PKC-{delta} is activated during apoptosis by caspase-mediated cleavage to become catalytically active (10) . This catalytic active fragment of PKC-{delta} translocates to the mitochondria to induce apoptotic events. Inhibition of PKC-{delta} blocks the disruption of the mitochondrial transmembrane potential (3 , 5) . These results indicate that PKC-{delta} plays an important role in triggering apoptosis through a mitochondrion-dependent pathway. However, the substrate of PKC-{delta} in the mitochondria remains unidentified.

The function of PLS is to translocate phospholipids bidirectionally between two compartments (11 , 12) . PLS1 translocates phospholipids between the inner and outer leaflets of the plasma membrane, which is asymmetric in lipid composition. Although PS is translocated to the outer leaflet during apoptosis, it is still unclear whether PLS1 is responsible for this translocation (13, 14, 15, 16, 17) . We studied another member of PLS family, PLS3, which is present in the mitochondria. Overexpression of PLS3 in the HEK293 cells enhanced apoptosis induced by UV irradiation, and blocking PLS3 with a dominant-negative mutant of PLS3 suppressed UV and etoposide-induced apoptosis.4 Here we report that PLS3 interacts with PKC-{delta} and is phosphorylated by PKC-{delta} with a very high affinity in vitro. Cells overexpressing PLS3 became apoptotic on treatment with phorbol ester and on expression of mitochondrial-targeted PKC-{delta}. These results indicate that PLS3 is the downstream target of PKC-{delta}.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Materials.
HEK293 and HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum. The cDNAs of PLS3 and PLS3(F258V) were cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) for expression in mammalian cells. PKC-{delta} was cloned into pCMV/Mito/myc vector (Invitrogen) to construct the mitochondrial targeted PKC-{delta}. Go6976, recombinant PKC-{delta} enzyme, and c-abl antibody were purchased from Calbiochem (San Diego, CA). Antibodies against PS, PT, PY, PKC-{delta}, and the PKC-{delta} inhibitor, rottlerin, were purchased from Sigma (St. Louis, MO).

In Vitro Phosphorylation and Kinetics Assay.
PLS3 protein was overexpressed as His-tagged protein by pQE (Qiagen) vector and then purified with nickel-column in 8 M urea. The PLS3 protein was eluted in urea and dialyzed against 2 M urea before usage. Immediately before in vitro phosphorylation, PLS3 was additionally diluted to <0.2 M urea in the final phosphorylation reaction. In vitro phosphorylation was performed with 0.1 µg of PKC-{delta}, {gamma}-[32P]ATP, and 1 µg of recombinant PLS3 protein in a reaction buffer as described in the manufacturer’s protocol (Calbiochem). The reaction mix was incubated at room temperature and stopped at various time points, or after 20 min in the kinetics study, with SDS loading buffer. The phosphorylated products were separated by SDS-PAGE and analyzed by autoradiography.

IP.
IP was performed with affinity purified PLS3 antibody at 1:100 dilution followed by Western analysis with PS, PT, PY, and PKC-{delta} antibodies. The Western blot was developed with chemiluminescence (Pierce, Rockford, IL).

Apoptotic Assays.
Apoptotic assays were performed with annexin V-PE (BD-PharMingen) per the manufacturer’s protocol. The TUNEL assay was performed according to the manufacturer’s protocol (Indianapolis, IN).

Subcellular Fractionation.
Subcellualr fractionation was performed by differential centrifugation. In brief, 107 cells were incubated in buffer containing 300 mM sucrose, 10 mM Tris (pH 7.5), 5 mM EDTA, 0.1% BSA, and 1 mM phenylmethylsulfonyl fluoride protease inhibitor for 5 min on ice. Cells were disrupted by passage through a 25-gauge needle or dounced for 10 strokes, and then were centrifuged at 1,000 x g for 5 min, 10,000 x g for 10 min, and 30,000 x g for 60 min, for collection of intact cells/nuclei, crude mitochondria, and microsomes, respectively. The final supernatants were used as cytosols. The mitochondria and the cytosolic fractions were analyzed for cytochrome c release with Western blot.

Immunofluorescent Staining.
Cells were fixed with 4% paraformaldehyde in PBS for 5 min. Cells were washed and permeabilized with 0.2% Triton X-100 in PBS for 2 min. Nonspecific reaction was blocked with 3% BSA for 15 min. Primary antibody against PKC-{delta} was used at 1:100, and secondary antibody with FITC was used at 1:200 dilution. The stained cells were visualized by Nikon fluorescence microscope Eclipse TE300.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
PLS3 Is a Phosphoprotein, and the Phosphorylation Increases after UV Irradiation.
To investigate the regulation of PLS3 activity, we studied the phosphorylation status of PLS3 during UV-induced apoptosis. We performed IP in HEK293 and HEK293-PLS3 cellular extracts with an antibody to PLS3, and probed the Western blot with antibodies specific for PS, PT, and PY. The immunoprecipitated PLS3 was recognized by the anti-PT antibody, but not by anti-PS or anti-PY (Fig. 1a)Citation , indicating that PLS3 is phosphorylated at the threonine residue. UV irradiation increased the PT signals in both the control HEK293 and HEK293-PLS3 cells. The same blot was probed with PLS3 antibody as a loading control, and revealed minimal difference in the amount of PLS3 protein before and after UV irradiation (Fig. 1a)Citation .



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  		Fig. 1. a, PLS3 is phosphorylated at threonine. HEK293 or HEK293-PLS3 cells were treated with or without UV. Cell lysates were immunoprecipitated with PLS3 antibody. Western blot analysis was then performed with PS, PT, PY, PLS3, and PKC-{delta} antibodies. b, UV irradiation induces apoptosis requires PKC-{delta}. HEK293 cells were treated with UV in the presence or absence of PKC-{delta} inhibitor, rottlerin (10 µM). Cells were harvested 4 h later, and fractionated to isolate mitochondria and cytosols. Fractions were analyzed with Western blot with cytochrome c, VDAC, and tubulin antibodies. In the bottom two panels, the mitochondrial fractions were additionally washed with mitochondrial isolation buffer to remove rottlerin. The washed mitochondria were then incubated with 100 nM PMA to activate PKC-{delta} to determine whether they were translocated to the mitochondria. After 20-min incubation, the mitochondria were separated from the supernatants. The supernatants and pellets were analyzed for cytochrome c.

 
PKC-{delta} Physically Interacts with PLS3.
We then looked for the kinase that phosphorylates PLS3. On the basis of the observation that PLS1 is phosphorylated by PKC-{delta} (9) , we studied whether PLS3 is the mitochondrial substrate of PKC-{delta}. We first reconfirmed that PKC-{delta} is essential for UV-induced apoptosis by blocking PKC-{delta} with a PKC-{delta} inhibitor, rottlerin, in our system. The HEK293 cells were fractionated, and the isolated mitochondria and cytosols were analyzed for cytochrome c release by Western blot. This study confirmed that cytochrome c was released to cytosol after UV irradiation, and this release was inhibited by rottlerin (Fig. 1b)Citation . The loading controls of the mitochondria or cytoplasms showed equal amounts of VDAC or tubulin. To examine the effect of translocated PKC-{delta} to mitochondrial cytochrome c release, we washed mitochondria with mitochondrial isolation buffer to remove rottlerin. The washed mitochondria were then treated with phorbol ester PMA to achieve maximal activation of PKC-{delta}, and the released cytochrome c after PMA treatment was studied. The mitochondria from non UV-irradiated cells had very minimal cytochrome c release, indicating very little PKC-{delta} present. The mitochondria from UV-irradiated but not rottlerin-treated cells released the most cytochrome c after PMA treatment, confirming the existence of PKC-{delta} in mitochondria after UV irradiation (Fig. 1Citation b, bottom two panels). The mitochondria from UV and rottlerin-treated cells released more cytochrome c than those from unirradiated cells, but less than those from UV irradiated cells, indicating that the amount of PKC-{delta} translocated to mitochondria decreased with rottlerin treatment. Therefore, there was a direct correlation between the amount of translocated PKC-{delta} and the released cytochrome c.

We then studied whether PLS3 interacted with PKC-{delta}. PLS3 was immunoprecipitated from the cell lysates of UV-irradiated and nonirradiated HEK293 cells. The precipitates were analyzed by immunoblotting for PLS3 and for PKC-{delta}. We observed PKC-{delta} in the blot, indicating that PLS3 and PKC-{delta} coimmunoprecipitate. Furthermore, this co-IP increased in cells after UV irradiation (Fig. 1Citation a, bottom panel). However, the amount of the PKC-{delta} that coimmunoprecipitated with PLS3 is only about 5–10% of total cellular PKC-{delta} based on the estimation from a PKC-{delta} blot for comparison (data not shown). This was not surprising, because only a portion of cellular PKC-{delta} translocates to mitochondria during apoptosis.

Because PKC-{delta} also interacts with c-abl kinase and phosphorylates c-abl (18) , we probed the same IP blot with c-abl antibody to determine whether PKC-{delta}, PLS3, and c-abl formed a complex. However, no c-abl signal was detected in the immunoprecipitates of PLS3 (data not shown). The fact that PLS3 was not phosphorylated at the tyrosine residue also argues against PLS3 as a substrate of c-abl kinase.

PKC-{delta} Phosphorylates PLS3 in Vitro.
Next we studied whether PLS3 is a substrate for PKC-{delta} using recombinant proteins. The PLS3 protein was a high-affinity substrate for PKC-{delta}, and the phosphorylation steadily increased over the first 25 min (Fig. 2a)Citation . We tested various concentrations of PLS3 and determined that the Km is 10.5 nM (Fig. 2, b and c)Citation . This is a very high affinity for PKC-{delta} toward PLS3 compared with other physiological substrates of the PKC family (2) .



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  		Fig. 2. Enzyme kinetics of PKC-{delta} phosphorylating PLS3. a, in vitro phosphorylation of PLS3 with recombinant PKC-{delta} in a time course from 0 to 25 min. Recombinant PLS3 protein (1 µg) and [{gamma}-32P]ATP were used as substrates, and recombinant PKC-{delta} was used as kinase for each in vitro phosphorylation reaction. b, kinetics study of PKC-{delta} toward PLS3 was performed using various concentrations of PLS3 and equal amount of PKC-{delta} for each reaction. The reaction was stopped after 20 min of incubation and analyzed by gel electrophoresis. The radioactivity of each PLS3 protein was determined by density analysis and plotted against the concentrations of the PLS3 protein. The plot in c is the double-reciprocal plot derived from the results to calculate the km value.

 
Phorbol Ester PMA Induces Apoptosis in Cells Overexpressing Wild-Type PLS3.
If PLS3 is a direct downstream effector of PKC-{delta} in the PKC-{delta}-induced apoptotic pathway, then overexpression of PLS3 might enhance apoptosis induced by PKC-{delta} activation. We incubated HeLa-PLS3 with phorbol ester PMA, and analyzed apo-ptosis with TUNEL assay and flow cytometry. PMA treatment of the control HeLa cells did not induce any apoptosis, but treatment of the HeLa-PLS3 cells shifted the curve to right (Fig. 3)Citation . To eliminate the effect of classic PKCs ({alpha}, ß, and {gamma}) that are also stimulated by PMA, we treated them with a combination of PMA and the indolocarbazole Go6976, which is a potent inhibitor for PKC-{alpha}, ß, and {gamma}, but does not affects PKC-{delta} (19) . The curve of HeLa-PLS3 cells had a similar shift to the right like that of PMA alone, indicating that this apoptotic effect by PMA is not related to the classic PKCs. These data support that the overexpression of the PKC-{delta} substrate PLS3 makes the mitochondrial effect of PKC-{delta} so dominant to induce apoptosis.



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  		Fig. 3. Overexpression of PLS3 converts PMA to a cell death agonist. HeLa-control, HeLa-PLS3, and HeLa-PLS3(F258V) cells were incubated with DMSO (black), 200 nM PMA (green), or the combination of 200 nM PMA and 200 nM Go6976 (red) for 2 h. Cells were then fixed with ethanol for TUNEL assays to quantify apoptosis.

 
We also constructed a mutant of PLS3 by mutating Phe258 to Val. This Phe258 is in a highly conserved calcium-binding motif, and mutation of the corresponding Phe at PLS1 completely abolishes the activity of PLS1 (20) . Overexpressing PLS3(F258V) mutant in HeLa cells resulted in a higher baseline apoptosis probably attributable to interfering with mitochondrial functions. PMA treatment of HeLa-PLS3(F258V) cells decreased apoptosis by shifting the curve to the left (Fig. 3Citation , bottom panel). When the same cells were treated with both PMA and Go6976, the left shift by PMA was inhibited, indicating that the left shift was because of activation of a survival signal from a classic PKC-related pathway.

Mitochondrial-targeted PKC-{delta} Induced Apoptosis in HeLa-PLS3 Cells.
Because PMA activates many other isoforms of PKCs and does not represent PKC-{delta}-specific stimulation, we constructed a mitochondrial-targeted PKC-{delta} by inserting the mitochondrial targeting sequence of cytochrome oxidase at the NH2 terminus of PKC-{delta}. The construct was transfected into HeLa cells, and G418-resistant clones were selected. No clones were obtained after 10 days of selection, whereas a control construct replacing PKC-{delta} with EGFP resulted in many clones with EGFP expressed in the mitochondria (data not shown). This indicates that overexpression of PKC-{delta} in the mitochondria is toxic to cells. We expressed the mitochondrial-targeted PKC-{delta} into HeLa-control, HeLa-PLS3, and HeLa-PLS3(F258V) cells, and studied apoptosis with annexin V-PE 36 h after transfection. Expression of the mitochondrial-targeted PKC-{delta} induced 17.3% of cell death in the control HeLa cells and 45.3% in HeLa-PLS3 cells, indicating that PLS3 enhanced the apoptotic effect of PKC-{delta} in the mitochondria. HeLa cells expressing PLS3(F258V) had only 24.9% of apoptosis in a similar study, far less effective in enhancing apoptosis than HeLa-PLS3 cells (Fig. 4a)Citation .



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  		Fig. 4. Overexpression of mitochondrial targeted PKC-{delta}. a, annexin V-PE study of the apoptosis was performed in the HeLa-control, HeLa-PLS3, and HeLa-PLS3(F258V) transfected with the mitochondrial-targeted PKC-{delta} construct or control vector. The percentages of apoptosis were indicated. b, immunofluorescent staining was performed in the vector-transfected control HeLa cells with PKC-{delta} antibody for colocalization with MitoTracker Red. c, HeLa cells were transfected with expression vector of the mitochondrial-targeted PKC-{delta}. Similar staining was performed with PKC-{delta} and MitoTracker Red. PKC-{delta} antibody staining (left panels); MitoTracker Red (middle panels); and overlay (right panels).

 
To prove that the PKC-{delta} was expressed in the mitochondria, we stained HeLa cells 3 days after transfection with PKC-{delta} antibody and colocalized with MitoTracker Red dye. In the vector-transfected cells, PKC-{delta} was diffusely present in the cytosol and perimembranous area, and did not overlay with MitoTracker Red (Fig. 4b)Citation . MitoTracker Red staining of the Mito-PKC-{delta}-transfected cells revealed all of the mitochondria clustered around the perinuclear area, a pattern reminiscent of early apoptosis. PKC-{delta} staining of the Mito-PKC-{delta}-transfected cells displayed more PKC-{delta} in the perinuclear area, which overlayed with the MitoTracker Red dye. This represents the overexpressed mitochondrial-targeted PKC-{delta}. There was also a less abundant cytosolic signal, likely from the endogenous PKC-{delta} (Fig. 4c)Citation .


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
We report here that PLS3 is the substrate of PKC-{delta} in the mitochondria. Overexpression of PLS3 sensitizes cells to apoptotic stimuli, presumably through augmentation of the effect of PKC-{delta}. This is best supported by the induction of apoptosis in HeLa-PLS3 cells with PMA. In the normal situation, PMA activates many different PKCs, which lead to activation of both cell survival and death signals. Depending on which pathway prevails, cells will become live or dead. When PLS3 is overexpressed, the PKC-{delta}-related PLS3 activation becomes dominant to induce apoptosis. This effect was not affected by a potent classic PKC inhibitor Go6976, confirming that it is indeed related with PKC-{delta}. The inactive PLS3(F258V) mutant failed to enhance the apoptotic effect of PKC-{delta}, and PMA actually protected HeLa-PLS3(F258V) cells through losing the downstream effector of apoptotic PKC-{delta} and activation of survival signals induced by PMA. This was confirmed by the addition of Go6976, which inhibited the survival effect of PMA.

Is PLS3 the only substrate of PKC-{delta} in the mitochondria? If so, blocking PLS3 with the dominant-negative mutant PLS3(F258V) would completely prevent apoptosis induced by the mitochondria-targeted PKC-{delta}, which appeared not to be the case. However, the fact that the cells expressing PLS3(F258V) had a high baseline cell death makes the interpretation difficult, because they may die from the overexpressed PKC-{delta} in the mitochondria or from poor mitochondrial functions. We cannot rule out the possibility that PKC-{delta} phosphorylates other substrates in mitochondria to induce apoptosis. Another question is whether PKC-{delta} is the only kinase that phosphorylates PLS3. The answer is likely negative as well. The fact that PLS3 has a baseline phosphorylation even before the induction of apoptosis (Fig. 1a)Citation indicates that PLS3 may be phosphorylated by other kinases, but the UV-enhanced phosphorylation is likely associated with additionally increased PLS3 activity. With the important role of PLS3 in mitochondria apoptosis, understanding the mechanism of PLS3 regulation is very important. PKC-{delta} is the kinase that contributes to the activation of PLS3.

It is interesting that both PLS1 and PLS3 are phosphorylated by PKC-{delta} during apoptosis but at different locations. Although the significance of PLS1 phosphorylation is unclear, it has been proposed that PLS1 might be related with the translocation of PS to the outer leaflet of plasma membrane. PLS3, present in the mitochondria, translocates cardiolipin to the outer membrane of the mitochondria, an interesting analogy when you consider that mitochondria are evolved from a prokaryote trapped inside of an eukaryote cell in the early stage of evolution. The PLS family is highly conserved in almost all of the eukaryotes, including yeast, Drosophila and Caenorhabditis elegans. Genetic studies in those lower organisms will be very helpful to dissect the mechanism of PLS regulation.


   ACKNOWLEDGMENTS
 
We thank Dr. Steve Prescott for reading the manuscript and helpful discussion.


   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 National Cancer Institute Grant K08CA795093, Howard Hughes Medical Institute Fellow to Faculty Transitional Program, and Huntsman Cancer Foundation. Back

2 To whom requests for reprints should be addressed, at Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Suite 5244, Salt Lake City, UT 84112. Phone: (801) 585-0611; Fax: (801) 585-0900; E-mail: ray.lee{at}hci.utah.edu Back

3 The abbreviations used are: PKC, protein kinase C; PLS, phospholipid scramblase; PS, phosphatidylserine; PT, phosphothreonine; PY, phosphotyrosine; PE, phycoerythrin; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; IP, immunoprecipitation; VDAC, voltage-dependent anion channel. Back

4 Q. Dai, Mutant phospholipid scramblase 3 blocks mitochondrial cardiolipin translocation and perturbs mitochondrial morphology and function, submitted for publication. Back

Received 9/30/02. Accepted 1/27/03.


   REFERENCES
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-{delta}-Induced Phosphorylation of Phospholipid Scramblase 3
Cancer Res., November 1, 2005; 65(21): 10016 - 10023.
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 J. Biol. Chem.Home page
A. E. Santiago-Walker, A. J. Fikaris, G. D. Kao, E. J. Brown, M. G. Kazanietz, and J. L. Meinkoth
Protein Kinase C {delta} Stimulates Apoptosis by Initiating G1 Phase Cell Cycle Progression and S Phase Arrest
J. Biol. Chem., September 16, 2005; 280(37): 32107 - 32114.
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 BloodHome page
P. Hampson, H. Chahal, F. Khanim, R. Hayden, A. Mulder, L. K. Assi, C. M. Bunce, and J. M. Lord
PEP005, a selective small-molecule activator of protein kinase C, has potent antileukemic activity mediated via the delta isoform of PKC
Blood, August 15, 2005; 106(4): 1362 - 1368.
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 Mol Cancer ResHome page
J. Liu, Q. Dai, J. Chen, D. Durrant, A. Freeman, T. Liu, D. Grossman, and R. M. Lee
Phospholipid Scramblase 3 Controls Mitochondrial Structure, Function, and Apoptotic Response
Mol. Cancer Res., October 1, 2003; 1(12): 892 - 902.
[Abstract] [Full Text] [PDF]

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