HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) An addition or correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kharbanda, S. Articles by Kufe, D. Search for Related Content PubMed PubMed Citation Articles by Kharbanda, S. Articles by Kufe, D. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 1, 322-327, January 7, 2000

Translocation of SAPK/JNK to Mitochondria and Interaction with Bcl-x_L in Response to DNA Damage^*

Surender Kharbanda^§¶, Satya Saxena^§, Kiyotsugu Yoshida, Pramod Pandey, Masao Kaneki, Qizhi Wang^**, Keding Cheng, Ying-Nan Chen^**, Angela Campbell^**, Thangrila Sudha, Zhi-Min Yuan, Jagat Narula, Ralph Weichselbaum, Carlo Nalin^**, and Donald Kufe

From the  Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115,  Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3EOV9, Canada, ^** Oncology Research Program, Preclinical Research, Novartis Pharmaceuticals Corp., East Hanover, New Jersey 07936, and  Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois 60637

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis. We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association of SAPK with the anti-apoptotic Bcl-x_L protein. SAPK phosphorylates Bcl-x_L on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-x_L, a mutant Bcl-x_L with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with Bcl-x_L in the apoptotic response to genotoxic stress.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The cellular response to ionizing radiation (IR)¹ and other genotoxic agents includes cell cycle arrest, activation of DNA repair and apoptosis, or physiological cell death. However, the intracellular signals that control these events are unclear. The available evidence indicates that IR induces these effects by direct interaction with DNA or through formation of reactive oxygen intermediates, which damage DNA and cell membranes (1). Whereas p53 has been shown to be involved in promoting apoptosis induced by IR exposure (2, 3), other studies have demonstrated that Bcl-2 and Bcl-x_L inhibit IR-induced apoptosis (4-6). The induction of apoptosis is associated with activation of aspartate-specific cysteine proteases (caspases) and cleavage of poly(ADP-ribose) polymerase, protein kinase C, and other proteins (7-9). The demonstration that cleavage of poly(ADP-ribose) polymerase and protein kinase C is blocked by Bcl-x_L has suggested that the Bcl-2-related family of anti-apoptotic proteins functions upstream to the activation of caspases (9-11).

The stress-activated protein kinases (SAPKs), also known as c-Jun amino-terminal kinases (JNKs) and p38 MAP kinase, are activated in response to diverse stimuli including DNA damage, heat shock, interleukin-1, tumor necrosis factor , and Fas (12-22). Recent studies have shown that activation of c-Abl by exposure to IR contributes to the induction of Jun kinase and p38 MAPK (14, 15, 22). Activation of SAPK and p38 MAPK pathways has been associated with induction of apoptosis (23-25). However, the mechanisms involved in SAPK/JNK-induced apoptosis are presently unclear. Recent studies have demonstrated that mitochondria play a central role in inducing apoptosis by releasing cytochrome c (26, 27). The demonstration that the anti-apoptotic Bcl-2 family of proteins is expressed in the mitochondrial membrane has also implicated mitochondria in the induction apoptosis (28, 29). Importantly, we and others have shown that overexpression of Bcl-2 or the related Bcl-x_L blocks the release of cytochrome c from mitochondria, which otherwise occurs when cells are signaled to undergo apoptosis (30-32).

The present studies demonstrate that exposure of U-937 cells to IR is associated with translocation of active SAPK to mitochondria and its association with the anti-apoptotic protein Bcl-x_L. The results also demonstrate that SAPK phosphorylates Bcl-x_L on threonines 47 and 115, and overexpression of mutant Bcl-x_L (A-47, A-115) causes a more potent inhibition of IR-induced apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cell Culture and Reagents-- Human U-937, U-937/Bcl-x_L, and U-937/Bcl-x_L(A-47,-115) myeloid leukemia cells 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. Rat-1/myc, Rat-1/myc/Bcl-x_L, and Rat-1/myc/Bcl-x_L(A-47,-115) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. IR was performed with a -ray source (Cs 173, Gamma Cell 1000, Atomic Energy of Canada, Ontario) at a fixed dose rate of 13 Gy/min. Cells were also treated with bleomycin (Sigma).

Immunofluorescence Microscopy-- Control and IR-treated U-937 cells immobilized on slides were fixed (3.7% formaldehyde), permeabilized (0.2% Triton X-100), and incubated with 20 ng/slide of anti-SAPK polyclonal antibody (Santa Cruz Biotechnology), followed by 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). Nuclei were stained with 4,6-diamidino-2-phenylindole (1 µg/ml in phosphate-buffered saline). 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).

Isolation of Cytoplasmic and Mitochondrial Fractions-- Cells were washed twice with phosphate-buffered saline, and cell fractionation was performed as described (30).

Immunoprecipitation and Immunoblot Analysis-- Immunoprecipitation was performed as described (33). In brief, soluble proteins were incubated with anti-SAPK (Santa Cruz) antibody for 1 h and precipitated with protein A-Sepharose for an additional 30 min. The resulting immune complexes were analyzed by immunoblotting with anti-Bcl-x antibodies (34). U-937/Bcl-xL cells were exposed to 20 Gy IR and harvested after 1 h. Total cell lysates were subjected to immunoprecipitation with anti-Bcl-xL, and the precipitates were analyzed by immunoblotting with anti-Bax (Santa Cruz) antibody.

Far Western Analysis-- Column-purified H₆-Bcl-x_LT, Bcl-x_ST, or H₆ proteins were resolved by SDS-PAGE and transferred to a nitrocellulose filter. The filters were incubated with purified GST-SAPK for 1 h, and immunoblot analysis was performed as described (30). Purified SAPK and H₆-SHPTP1 proteins were resolved by SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with H₆-Bcl-x_L or H₆ proteins and analyzed by immunoblotting with anti-Bcl-x_L antibody.

Immune Complex Kinase Assays-- GST-SAPK or anti-SAPK immunoprecipitates from cells were incubated with H₆-Bcl-x_LT, Bcl-x_ST, H₆-Bcl-x_LT(A-47,-115), or GST-Jun (35), and in vitro immune complex kinase assays were performed as described (15).

Phosphopeptide Mapping and Phosphoamino Acid Analysis-- ³²P-Labeled Bcl-x_LT recovered from unfixed dried SDS-polyacrylamide gels by homogenizing gel slices in the presence of trichloroacetic acid was oxidized with performic acid, resuspended in 25 µl of 50 mM NH₄HC0₃, and digested overnight at 37 °C with 10 µg of tosylphenylalanyl chloromethyl ketone-treated trypsin (36). After digestion, samples were repeatedly lyophilized. Tryptic digests of the phosphorylated Bcl-x_LT were subjected to electrophoresis using pH 1.9 buffer (15% acetic acid, 5% formic acid) followed by ascending chromatography with Scheidtmann buffer (isobutyric acid:pyridine:acetic acid:butanol:water (65:5:3:2:29)) as described (37). Phosphopeptides were visualized by autoradiography. Individual spots were scraped from the cellulose plates, and phosphopeptides were eluted with pH 1.9 buffer. After lyophilization, phosphopeptides were subjected to partial acid hydrolysis, and hydrolized products were subjected to two-dimensional electrophoresis.

Phosphorylation of Bcl-x_L by SAPK in Vivo-- 293T cells were transiently cotransfected with HA-Bcl-x_L and MEKK-1 or MEKK-1 K-N by LipofectAMINE (Life Technologies, Inc.). At 36 h after transfection, cells were labeled with [³²P]orthophosphate (0.5 mCi/ml) in a phosphate-free media for 3 h. Total cell lysates were then subjected to immunoprecipitation with anti-HA antibody. Immunopurified labeled HA-Bcl-xL under conditions of active or inactive MEKK-1 were separated by SDS-PAGE and analyzed by autoradiography. Anti-HA immunoprecipitates were also analyzed by immunoblotting with anti-HA. As a control, total cell lysates from transfected but unlabeled 293T cells were subjected to immunoprecipitation with anti-SAPK, and in vitro immune complex kinase assays were performed using GST-Jun as substrate as described above.

Generation of Bcl-x_L and Bcl-x_LT Mutants-- Mutations in full-length (Bcl-x_L) or truncated (Bcl-x_LT) and generation of various plasmids (HA-Bcl-x_L) were performed as described (30).

Cell Survival and Apoptosis Assays-- U-937, U-937/Bcl-x_L, or U-937/Bcl-x_L(A-47,-115) cells were treated with 20 Gy IR and harvested at the indicated times. Terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) assays were performed as described (38). Numbers of cells with sub-G₁ DNA content were determined with a ModFit LT program (Verity Software House) (38). Rat-1/myc, Rat-1/myc/Bcl-x_L, or Rat-1/myc/Bcl-x_L(A-47,-115) cells were treated with bleomycin. After 3 days, apoptotic cells were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays as described (39, 40).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The stress-activated protein kinase (SAPK/JNK) is induced by IR and other genotoxic agents (13, 14, 16, 41-43). Other studies support a role for SAPK in induction of apoptosis (23, 24). Translocation of activated SAPK to the nucleus has been found in hypoxic cells and after treatment with ultraviolet radiation (44, 45). Here we investigated subcellular localization of SAPK in response to genotoxic stress by measuring intracellular fluorescence with a high sensitivity CCD camera and image analyzer. Examination of the distribution of fluorescence markers in control U-937 cells showed distinct patterns for anti-SAPK (red signal) and a mitochondrion-selective dye (Mitotracker; green signal) (Fig. 1A). By contrast, exposure to IR was associated with a dramatic change in fluorescence signals (red and green  yellow/orange), supporting translocation of SAPK to mitochondria (Fig. 1A). To confirm these findings, cytoplasmic and mitochondrial fractions were subjected to immunoblotting with anti-SAPK. Consistent with the immunofluorescence data, IR exposure induced translocation of SAPK to mitochondria (Fig. 1B). Purity of the mitochondrial and cytoplasmic fractions was confirmed by reprobing the blots with an antibody against the mitochondrial-specific HSP60 protein (Fig. 1B). These findings demonstrate that IR induces SAPK to translocate to mitochondria.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Translocation of SAPK to mitochondria in response to IR. A, U-937 cells were treated with 20 Gy IR and harvested after 1 h. After washing, cells were immobilized on slides, fixed, and incubated with anti-SAPK (Santa Cruz) antibody followed by Texas Red-conjugated goat anti-rabbit IgG. Mitochondria were stained with the mitochondria-selective permeant dye Mitotracker Green FM. Nuclei were stained with 4,6-diamidino-2-phenylindole. The slides were visualized using a Zeiss Auxiphot fluoresence microscope coupled to a high sensitivity CCD camera and image analyzer. Red signal, SAPK; green signal, Mitotracker; yellow/orange signal, co-localization of SAPK and mitochondria signals. B, U-937 cells were treated with 20 Gy IR and harvested at 1 h. Cytoplasmic (Cyto) and mitochondrial (Mito) fractions (34) were isolated and subjected to immunoblotting with anti-SAPK or anti-HSP60 (Sigma) antibodies.

Bcl-x_L is predominantly a mitochondrial protein (29, 46). To determine if SAPK associates with Bcl-x_L following translocation to mitochondria, we studied co-localization of these proteins using immunofluorescence microscopy. In unirradiated cells, staining patterns of SAPK (green signal) and Bcl-x_L (red signal) were mainly distinct (Fig. 2A). Importantly, IR exposure was associated with apparent co-localization of these proteins, as evident from the marked changes of fluorescence signals to yellow/orange (Fig. 2A). To determine biochemically whether SAPK forms a complex with Bcl-x_L, we subjected anti-SAPK immunoprecipitates to immunoblotting with anti-Bcl-x. Cytoplasmic fractions from control and irradiated cells demonstrated little if any association of SAPK and Bcl-x_L (Fig. 2B); however, a similar analysis of the mitochondrial fraction demonstrated that IR induces binding of SAPK to Bcl-x_L (Fig. 2B). To address whether Bcl-x_L interacts directly with SAPK, we prepared a truncated His-tagged Bcl-x_L protein lacking 21 amino acids from the carboxyl terminus to avoid aggregation and precipitation in in vitro reactions (H₆-Bcl-x_LT) (30). GST-SAPK fusion protein was resolved by gel electrophoresis and transferred to a nitrocellulose filter. Two identical filters were prepared. Filters were separately incubated either with H₆-Bcl-x_L or H6 proteins and analyzed by immunoblotting with anti-Bcl-x_L. As a control, purified H₆-SHPTP1 protein was also resolved by SDS-PAGE and transferred to nitrocellulose filters. After incubation with H₆-Bcl-x_LT, the filters were probed with anti-Bcl-x_L. By contrast to SHPTP1, reactivity of anti-Bcl-x_L at the position corresponding to GST-SAPK (~80 kDa) indicated direct interaction between Bcl-x_L and SAPK (Fig. 2C). In the reciprocal experiment, Bcl-x_LT and Bcl-x_ST were resolved by gel electrophoresis and transferred to filters. After incubation with recombinant GST-SAPK, the filters were probed with anti-SAPK. The results confirmed direct binding of SAPK to Bcl-x_L and not Bcl-x_S (Fig. 2D).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Association of SAPK with Bcl-xL. A, U-937 cells were treated with 20 Gy IR and harvested after 1 h. Cells were incubated with anti-SAPK and anti-Bcl-xL followed by fluorescein isothiocyanate-conjugated donkey anti-goat IgG and Texas Red-conjugated goat anti-rabbit IgG antibodies. Red signal, Bcl-xL; green signal, SAPK; yellow/orange signal, co-localization of Bcl-xL and SAPK. top panels, 100×; bottom panels, 40×. B, U-937 cells were exposed to 20 Gy IR and harvested after 1 h. Cytoplasmic (Cyto) and mitochondrial (Mito) lysates from control, and IR-treated U-937 cells were subjected to immunoprecipitation (IP) with anti-SAPK antibodies. The proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting (IB) with anti-Bcl-xL antibodies. Mitochondrial lysate from U-937/Bcl-xL cells was used as a positive (+ve) control. C, recombinant purified GST-SAPK protein was subjected to 10% SDS-PAGE and transferred to nitrocellulose filters (left panel). H6-SHPTP1 (SHPTP) protein was included as a control (right panel). The filters were incubated with H6-Bcl-xLT or H6 proteins and analyzed by immunoblotting with anti-Bcl-xL antibodies. D, nickel-purified H6-Bcl-xLT, H6, or GST-Bcl-xST proteins were separated by 12.5% SDS-PAGE and transferred to nitrocellulose. The filters were then incubated with purified recombinant GST-SAPK protein and analyzed by immunoblotting with anti-SAPK.

SAPK phosphorylates c-Jun in response to genotoxic stress. To determine whether Bcl-x_LT is also a substrate for SAPK phosphorylation, recombinant H₆-Bcl-x_LT was incubated with GST-SAPK in the presence of [-³²P]ATP. Analysis of the reaction products demonstrated that, like GST-Jun, SAPK phosphorylates H₆-Bcl-x_LT (Fig. 3A). Previous studies have shown that SAPK binds and phosphorylates c-Jun (47, 48). In addition, deletion of the SAPK-binding site in c-Jun is associated with a marked decrease in SAPK-mediated phosphorylation of c-Jun (12). In concert with the absence of SAPK binding to Bcl-x_S, there was little if any phosphorylation of GST-Bcl-x_ST by SAPK (Fig. 3A). As SAPK preferentially phosphorylates serine and/or threonine residues that are followed by prolines, we asked if the two Thr-Pro (amino acids 47, 48 and 115, 116) and/or one Ser-Pro (amino acids 62, 63) sites in Bcl-x_L are phosphorylated by SAPK. Phosphopeptide mapping studies of Bcl-x_L phosphorylated by SAPK demonstrated the presence of two ³²P-labeled peptides (Fig. 3B). Phosphoamino acid analysis of these peptides revealed phosphorylation on Thr residues (Fig. 3B). These studies identified Thr-47 and Thr-115 as SAPK phosphorylation sites. Accordingly, there was no detectable SAPK-mediated phosphorylation of a Bcl-x_LT mutant in which these sites had been mutated to alanines (Ala-47,Ala-115) (Fig. 3C).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of Bcl-xLT by SAPK. A, the indicated amounts of purified H6-Bcl-xLT protein were incubated with purified GST-SAPK with [-32P]ATP and kinase buffer at 30 °C for 15 min (left panel). GST-Jun (13) protein was used as a positive control. Purified GST-SAPK protein was also incubated separately with GST-Bcl-xST (right panel). Kinase reactions were terminated by adding SDS buffer and boiling for 5 min. The proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. B, phosphorylated Bcl-xLT protein recovered from homogenized slices of the SDS-polyacrylamide gel were digested with trypsin and then separated by electrophoresis at pH 1.9 (horizontal dimension with anode to the left) followed by ascending chromatography. For phosphoamino acid analysis, individual spots were scraped from cellulose plates, and phosphopeptides (a and b) were eluted with pH 1.9 buffer. After lyophilization, phosphopeptides were subjected to partial acid hydrolysis, and products were subjected to two-dimensional electrophoresis as described. PT, phosphothreonine; PS, phosphoserine; PY, phosphotyrosine. C, purified H6-Bcl-xLT or H6-Bcl-xLT(A-47,-115) were incubated with purified GST-SAPK and [-32P]ATP in kinase buffer for 15 min at 30 °C. GST-Jun protein was used as a positive control. The proteins were separated by 10% SDS-PAGE and analyzed by autoradiography.

To determine whether SAPK phosphorylates Bcl-x_L in vivo, 293T cells were transiently cotransfected with HA-Bcl-x_L and MEKK-1 (49) (an upstream regulator of SAPK activity) (12, 50, 51) or the kinase-inactive MEKK-1 K-N mutant. Cells were labeled with [³²P]orthophosphate for 3 h. After transfection and labeling, total cell lysates were subjected to immunoprecipitation with anti-HA, and the precipitates were analyzed by autoradiography. The results demonstrate that, in contrast to MEKK-1, phosphorylation of Bcl-x_L is significantly inhibited in cells that overexpress MEKK-1 K-N (Fig. 4A). As a control, in vitro SAPK immune complex kinase assays were performed using GST-Jun as substrate. The results demonstrate that, in concert with the previous findings (12, 50, 51), overexpression of MEKK-1, but not MEKK-1 K-N, is associated with activation of SAPK (Fig. 4B and data not shown). Taken together, these findings indicated that SAPK phosphorylates Bcl-x_L both in vitro and in vivo.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of Bcl-xL by SAPK in vivo. A, 293T cells were transiently transfected with 5 µg HA-Bcl-x and wild-type MEKK-1 or MEKK-1 K-N. Cells were metabolically labeled with [32P]orthophosphate for 3 h. Total cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibody. Immunopurified labeled HA-Bcl-xL was separated by SDS-PAGE and analyzed by autoradiography (upper panel). Anti-HA immunoprecipitates were also analyzed by immunoblotting (IB) with anti-HA (lower panel). B, 293T cells were transiently transfected with HA-Bcl-xL and MEKK-1 or MEKK-1 K-N. Total cell lysates were subjected to immunoprecipitation with anti-JNK, and in vitro immune complex kinase assays were performed using GST-Jun as substrate.

Since activation of SAPK contributes to induction of apoptosis (23, 24), we asked if the Bcl-x_L(A-47,-115) mutant is functional in regulating the apoptotic response. Sub-G₁ DNA content in propidium iodide-stained cells was assessed as a measure of apoptosis. Although exposure of U-937 cells to IR increased the proportion of cells with sub-G₁ DNA, expression of the Bcl-x_L(A-47,-115) mutant resulted in a smaller sub-G₁ peak and increased resistance to apoptosis compared with that in U-937 cells expressing wild-type Bcl-x_L (Fig. 5A). To extend these findings, we used Rat-1 cells transformed with c-myc (Rat-1/myc) that respond to genotoxic stress with induction of apoptosis (46). Overexpression of wild-type Bcl-x_L blocked bleomycin-induced cell death of Rat-1/myc cells (Fig. 5B). Significantly, overexpression of the Bcl-x_L(A-47,-115) mutant was more effective than Bcl-x_L in blocking induction of apoptosis (Fig. 5B). Similar results were obtained in four independently selected Rat-1/myc clones that express Bcl-x_L(A-47,-115) (Fig. 5B). Thus, interaction between SAPK and Bcl-x_L is functionally important in induction of apoptosis in different cell types treated with diverse genotoxic agents.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   DNA damage-dependent inhibition of apoptosis in cells stably overexpressing Bcl-xL(A-47,-115). A, U-937, U-937/Bcl-xL, or U-937/Bcl-xL(A-47,-115) cells were treated with 20 Gy IR and harvested at the indicated times. After fixing, cells were stained with propidium iodide, and sub-G1 DNA content was measured using FACScan (upper panel). The percentage of apoptotic cells with sub-G1 DNA content is expressed as the mean ± S.D. from three independent experiments performed in duplicate for the U-937 (hatched bar), U-937/Bcl-xL (open bar), and U-937/Bcl-xL(A-47,-115) (solid bar) cells (lower panel). The percentage of the following cells in G0/G1, S, G2/M phases for were: U-937, 72.5, 25.2, 2.2%; U-937/Bcl-xL, 57.4, 37.0, 5.5%; U-937/Bcl-xL (A-47,-115), 61.3, 32.6, 6.1%. B, Rat-1/myc (wild-type (wt)), Rat-1/myc/Bcl-xL, or Rat-1/myc/Bcl-xL(A-47,-115) (four independently selected clones) cells were plated in 96-well tissue culture dishes and treated 12 h later with bleomycin. After 3 days, the percentage apoptotic cells were determined. Results are expressed as the mean ± S.D. from three independent determinations each done in triplicate.

Previous studies have shown that Bcl-x_L heterodimerizes with Bax (52). Other studies have demonstrated that Bad selectively forms heterodimers with Bcl-x_L (53). When Bad heterodimerizes with Bcl-x_L in mammalian cells, it displaces Bax from Bcl-x_L and promotes cell death (53). To determine whether phosphorylation of Bcl-x_L by SAPK affects the interaction of Bax with Bcl-x_L, U-937 cells overexpressing Bcl-x_L were exposed with IR, and total cell lysates were subjected to immunoprecipitation with anti-Bcl-x_L. The protein precipitates were then analyzed by immunoblotting with anti-Bax. The results demonstrate that SAPK-mediated phosphorylation of Bcl-x_L has no detectable effect on its association with Bax (data not shown).

SAPK is activated by genotoxic stress (13, 14, 16, 41-43) and functions upstream to induction of apoptosis in response to DNA damage (23, 24). The present work demonstrates for the first time that genotoxic stress induces translocation of SAPK to mitochondria. Recent studies have supported the release of cytochrome C from mitochondria to the cytosol in the apoptotic response to DNA damage (30). However, the initial signal that triggers mitochondrial changes in response to apoptotic stimuli is presently not known. The findings that Bcl-2 and Bcl-x_L block release of cytochrome C and activation of caspases has further supported the importance of mitochondria in induction of apoptosis (30-32). In this context, our finding that SAPK associates with Bcl-x_L in mitochondria provides a potential link between mitochondrial translocation of SAPK and apoptosis. The results demonstrate that SAPK phosphorylates Bcl-x_L at Thr-Pro sites. Thr-47 resides in a 60-residue loop that is nonessential for anti-apoptotic activity (54, 55), whereas Thr-115 is adjacent to the 3 helix, which may be important structurally for formation of ion channels. The Bcl-x_L(A-47,-115) mutant was more effective than wild-type Bcl-x_L in blocking apoptosis. Thus, the Bcl-x_L mutant may, as a defective substrate, promote formation of stable SAPK complexes that would otherwise dissociate after phosphorylation of wild-type Bcl-x_L. In this regard, our results demonstrate that the Thr to Ala mutant of Bcl-x_L associates with SAPK with higher affinity than that obtained with wild-type Bcl-x_L (data not shown). Whereas SAPK activation is necessary for induction of apoptosis (23, 24), sequestration of SAPK in mitochondria by Bcl-x_L(A-47,-115) could abrogate other functions of SAPK, particularly in the cytoplasm or nucleus, that are required for the apoptotic response.

	ACKNOWLEDGEMENTS

We thank J. Kyriakis and L. Zon for various SAPK cDNA constructs, L. Boise and C. Thompson for anti-Bcl-x antibodies, and Andrew Place, Atsuko Nakazawa, and Xiangquo Qui for technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA 75216 (to S. K.) and CA55421 (to D. K.) and by a Medical Research Council of Canada Grant MT-14361 (to S. S.).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: Dept. of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2938; Fax: 617-632-2934; E-mail: surender_kharbanda@dfci.harvard.edu.

^¶ These authors contributed equally.

	ABBREVIATIONS

The abbreviations used are: IR, ionizing radiation; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; Bcl-2, B-cell lymphoma protein-2; PAGE, polyacrylamide gel electrophoresis; MEKK-1, mitogen/extracellular-regulated kinase kinase-1; Gy, gray; GST, glutathione S-transferase; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				B. Saberi, M. Shinohara, M. D. Ybanez, N. Hanawa, W. A. Gaarde, N. Kaplowitz, and D. Han Regulation of H2O2-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes Am J Physiol Cell Physiol, July 1, 2008; 295(1): C50 - C63. [Abstract] [Full Text] [PDF]

				N. Hanawa, M. Shinohara, B. Saberi, W. A. Gaarde, D. Han, and N. Kaplowitz Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury J. Biol. Chem., May 16, 2008; 283(20): 13565 - 13577. [Abstract] [Full Text] [PDF]

				X. Chen, M. Rzhetskaya, T. Kareva, R. Bland, M. J. During, A. W. Tank, N. Kholodilov, and R. E. Burke Antiapoptotic and Trophic Effects of Dominant-Negative Forms of Dual Leucine Zipper Kinase in Dopamine Neurons of the Substantia Nigra In Vivo J. Neurosci., January 16, 2008; 28(3): 672 - 680. [Abstract] [Full Text] [PDF]

				A. Kauskot, F. Adam, A. Mazharian, N. Ajzenberg, E. Berrou, A. Bonnefoy, J.-P. Rosa, M. F. Hoylaerts, and M. Bryckaert Involvement of the Mitogen-activated Protein Kinase c-Jun NH2-terminal Kinase 1 in Thrombus Formation J. Biol. Chem., November 2, 2007; 282(44): 31990 - 31999. [Abstract] [Full Text] [PDF]

				Y. Kim, P. Zhou, L. Qian, J.-Z. Chuang, J. Lee, C. Li, C. Iadecola, C. Nathan, and A. Ding MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival J. Exp. Med., September 3, 2007; 204(9): 2063 - 2074. [Abstract] [Full Text] [PDF]

				H. Malhi, F. J Barreyro, H. Isomoto, S. F Bronk, and G. J Gores Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity Gut, August 1, 2007; 56(8): 1124 - 1131. [Abstract] [Full Text] [PDF]

				A. Autret, S. Martin-Latil, L. Mousson, A. Wirotius, F. Petit, D. Arnoult, F. Colbere-Garapin, J. Estaquier, and B. Blondel Poliovirus Induces Bax-Dependent Cell Death Mediated by c-Jun NH2-Terminal Kinase J. Virol., July 15, 2007; 81(14): 7504 - 7516. [Abstract] [Full Text] [PDF]

				T. H. Tran, P. Andreka, C. O. Rodrigues, K. A. Webster, and N. H. Bishopric Jun Kinase Delays Caspase-9 Activation by Interaction with the Apoptosome J. Biol. Chem., July 13, 2007; 282(28): 20340 - 20350. [Abstract] [Full Text] [PDF]

				S. A. Lee and M. Jung The Nucleoside Analog Sangivamycin Induces Apoptotic Cell Death in Breast Carcinoma MCF7/Adriamycin-resistant Cells via Protein Kinase C{delta} and JNK Activation J. Biol. Chem., May 18, 2007; 282(20): 15271 - 15283. [Abstract] [Full Text] [PDF]

				M. Wilhelm, Z. Xu, N. V. Kukekov, S. Gire, and L. A. Greene Proapoptotic Nix Activates the JNK Pathway by Interacting with POSH and Mediates Death in a Parkinson Disease Model J. Biol. Chem., January 12, 2007; 282(2): 1288 - 1295. [Abstract] [Full Text] [PDF]

				G. Kroemer, L. Galluzzi, and C. Brenner Mitochondrial Membrane Permeabilization in Cell Death Physiol Rev, January 1, 2007; 87(1): 99 - 163. [Abstract] [Full Text] [PDF]

				N. Lopez-Sanchez, J.-R. Rodriguez, and J. M. Frade Mitochondrial c-Jun NH2-Terminal Kinase Prevents the Accumulation of Reactive Oxygen Species and Reduces Necrotic Damage in Neural Tumor Cells that Lack Trophic Support Mol. Cancer Res., January 1, 2007; 5(1): 47 - 60. [Abstract] [Full Text] [PDF]

				M. A. Bogoyevitch and B. Kobe Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 1061 - 1095. [Abstract] [Full Text] [PDF]

				G. De Chiara, M. E. Marcocci, M. Torcia, M. Lucibello, P. Rosini, P. Bonini, Y. Higashimoto, G. Damonte, A. Armirotti, S. Amodei, et al. Bcl-2 Phosphorylation by p38 MAPK: IDENTIFICATION OF TARGET SITES AND BIOLOGIC CONSEQUENCES J. Biol. Chem., July 28, 2006; 281(30): 21353 - 21361. [Abstract] [Full Text] [PDF]

H. Malhi, S. F. Bronk, N. W. Werneburg, and G. J. Gores Free Fatty Acids Induce JNK-dependent Hepatocyte Lipoapoptosis J. Biol. Chem., April