The tyrosine kinase Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expression

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Abstract

Tyrosine kinases of the Src family have been implicated in key biological processes. Here, we provide evidence that p56Lck, a lymphoid-specific Src kinase, is involved in the activation of the mitochondrial apoptosis pathway. Lck-deficient T cells were completely resistant to anticancer drugs. In contrast, apoptosis sensitivity to death receptors was not altered, indicating a specific interference of Lck with the mitochondrial pathway. Re-expression of Lck restored sensitivity to drug-induced apoptosis and triggered mitochondrial cytochrome c release and caspase activation. Further analysis identified that the sensitization by Lck was independent of classical mediators of T-cell signaling, but essentially involved the Bcl-2 protein Bak. Expression of Bak was completely absent in Lck-deficient cells, while re-expression of Lck transcriptionally triggered Bak expression and conferred sensitivity to apoptosis, associated with a proapoptotic conformational change of Bak. Furthermore, in vitro the truncated fragment of Bid specifically activated Bak and cytochrome c release only from mitochondria of Lck-expressing cells. These results do not only demonstrate a sentinel role of Lck in drug resistance but also delineate a hitherto unknown pathway of Src kinases in regulation of Bcl-2 proteins.

Introduction

Nonreceptor protein tyrosine kinases exert crucial roles in a variety of cellular processes including cell growth, differentiation, apoptosis and gene transcription. Tyrosine kinases normally function as tightly regulated switches in the signal transduction network of the cell. If these switches become stuck in the ‘on’ position, they have the potential to induce oncogenic transformation (Skorski, 2002). In T-lymphocytes, protein tyrosine kinases play an integral role in the activation of cells through various immunoreceptor molecules, such as the T-cell receptor (TCR)/CD3 complex, CD2, LFA-1 and MHC class I (Singer and Koretzky, 2002; Zamoyska et al., 2003). Engagement of the TCR leads to a rapid rise in intracellular protein tyrosine phosphorylation, followed by a series of biochemical events that result in effector function in T cells. An initial TCR signal is mediated by the activation of the Src family tyrosine kinases Lck and Fyn, which phosphorylate specialized motifs in the signal-transducing subunits of the TCR complex. Phosphorylation of these immunoreceptor tyrosine-based activation motifs creates docking sites for another tyrosine kinase, ZAP-70. As a result of ZAP-70 recruitment, the TCR is effectively endowed with kinase function, leading to the activation of a series of second-messenger cascades (Kane et al., 2000). These include, among others, phospholipase C, the phosphatidylinositol pathway, GTPases, MAP kinase cascades and proinflammatory transcription factors.

A few recent studies indicate that tyrosine kinases might not only play a role in T-cell activation but also in apoptosis. During activation-induced cell death, Lck has been implicated in the induction of CD95 ligand expression (Gonzalez-Garcia et al., 1997). Furthermore, studies using Lck-deficient T-cell lines have suggested that Lck eventually plays a role in transducing signals leading to apoptosis in response to irradiation, ceramide and HIV-Tat exposure (Belka et al., 1999; Manna et al., 2000; Manna and Aggarwal, 2000; Hur et al., 2004). Likewise, the B-cell receptor-associated kinases Btk and Lyn have been implicated in apoptosis in response to radiation and growth factor deprivation (Uckun et al., 1996; Qin et al., 1997; Hernandez-Hansen et al., 2004). The mechanism of how activation of Lck or related protein kinases contributes to apoptosis in certain situations, while inducing proliferation in others, is unknown.

Apoptotic signaling cascades can be divided into two major pathways, both of which depend on the formation of large multiprotein complexes and the activation of caspases (Schulze-Osthoff et al., 1998; Schwerk and Schulze-Osthoff, 2005). Upon formation of the death-inducing signaling complex in the extrinsic death receptor pathway or the apoptosome in the intrinsic mitochondrial pathway, caspase-8 or -9 is proteolytically processed resulting in the activation of downstream caspases and cleavage of numerous substrates (Fischer et al., 2003; Schwerk and Schulze-Osthoff, 2003). Intrinsic pathway stimuli, including many agents that elicit cellular stress, require the release of mitochondrial proteins, such as cytochrome c and Smac/Diablo, for caspase activation, whereas extrinsic pathway signals activate caspases more directly. Bcl-2 proteins function at a critical checkpoint in the intrinsic pathway to regulate mitochondrial membrane permeability and release of cytochrome c (Ferri and Kroemer, 2001).

Two major groups of Bcl-2 family proteins exist: the prosurvival members such as Bcl-2, Bcl-xL, Bfl-1/A1, Bcl-w, Mcl-1 and Boo, and the proapoptotic members including Bax, Bak and Bok (Cory et al., 2003; Daniel et al., 2003). Three-dimensional imaging suggests that both pro- and antiapoptotic Bcl-2 family members share a common structure including three or four regions, called the Bcl-2 homology (BH) domains. Bcl-2 proteins interact with another group of proapoptotic proteins that share at least one common motif with Bcl-2, namely the BH3 domain. BH3-only members include, for instance, Bad, Bim, Bid, Bik/Nbk, Puma and Noxa (Puthalakath and Strasser, 2002). Some BH3-only proteins selectively interact with antiapoptotic Bcl-2 family members, whereas others also interact with proapoptotic members. BH3-only proteins have been proposed to act as allosteric regulators of the Bcl-2 proteins and as sensors of apoptotic signaling. For example, during death receptor-mediated apoptosis Bid is cleaved into the death-promoting p15 fragment, tBid, which is targeted to mitochondria and binds to either Bax or Bak, resulting in their activation.

The generation of Bax and Bak knockout mice has elucidated how proapoptotic Bcl-2 proteins could induce cell death. Cells lacking both Bax and Bak do not die in response to various apoptotic stimuli including BH3-only proteins, in contrast to cells expressing either one of these molecules (Wei et al., 2001). This provided the insight that BH3-only proteins require at least one Bax-type partner to induce cell death. Both Bax and Bak undergo a conformational change in response to apoptotic stimuli, which mediates exposure of their occluded N-terminus (Desagher et al., 1999; Griffiths et al., 1999). Moreover, they assemble into homomultimers with channel-forming properties in the mitochondrial membrane (Antonsson et al., 1997; Wei et al., 2000). The conformational change of Bak or Bax is inducible by BH3-only proteins and inhibitable by Bcl-2. The model emerging from these findings is that cytochrome c release depends on interaction between a BH3-only protein and a Bax-type partner, which allows formation of a Bax-type transmembrane pore. Bak and Bax are generally assumed to be able to substitute for each other, since deficiency for both genes is required to render cells completely resistant to apoptosis. However, recent evidence also suggests that Bax and Bak exert nonredundant roles and differentially regulate cell death (Panaretakis et al., 2002; Cartron et al., 2003; Gillissen et al., 2003; Klee and Pimentel-Muinos, 2005). Bax and Bak also differ in their initial cellular location. While Bak is constitutively integrated into the ER or outer mitochondrial membrane, Bax typically resides in the cytosol or is only loosely associated with membrane surfaces (Wolter et al., 1997).

In the present study, we investigated the role of the Lck kinase in apoptosis. We found that Lck-deficient Jurkat T cells were completely resistant to mitochondrial apoptosis triggered by anticancer drugs, whereas the sensitivity to the death receptor pathway was largely unaffected. Furthermore, we identified that the block of apoptosis by Lck deficiency was caused by the selective failure to express the proapoptotic protein Bak, even though the cells remained proficient for Bax. Re-expression of Lck reconstituted Bak expression, caspase activation and, moreover, restored the capability of purified mitochondria to release cytochrome c in response to Bid treatment. Thus, our results identify signal transduction by Lck kinase as a novel pathway that controls Bak expression. Despite overlapping functions of Bak and Bax, our results also show that Lck-controlled Bak expression is required for the mitochondrial apoptotic pathway and thus may contribute to chemoresistance of human leukemic cells.

Results

Deficiency of the Lck tyrosine kinase results in resistance to apoptosis induced by anticancer drugs

T-cells express primarily two members of the Src family of tyrosine kinases, Lck and Fyn, both of which have been implicated in various biological processes. To examine the role of Lck in apoptosis, we used the JCaM1.6 cell line, a genetic variant of Jurkat T cells deficient in p56Lck protein because of a deletion of exon 7 encoding its ATP binding site (Straus and Weiss, 1992). As revealed by immunoblot analysis, an antibody against the N-terminus of Lck detected small amounts of a 49 kDa splice product in JCaM cells (Figure 1a), whereas an antibody against the C-terminus did not show any immunoreactivity (data not shown). Both the parental Jurkat cells and Lck-transfected JCaM cells expressed similar amounts of the wild-type kinase at 56 kDa. Analysis of apoptosis showed that, in contrast to parental Jurkat cells, JCaM cells were almost completely resistant to apoptosis induced by the topoisomerase II inhibitor etoposide, even at high drug concentrations and after prolonged incubation for 48 h (Figure 1b). Apoptosis resistance of JCaM cells was not only observed after treatment of cells with a topoisomerase inhibitor but also after incubation with unrelated anticancer drugs including doxorubicin, daunorubicin and mitomycin C. Interestingly, re-expression (Figure 1b) of Lck in JCaM cells fully overcame drug resistance and reconferred apoptosis sensitivity in response to all chemotherapeutic agents.

Figure 1
figure1

Lck-deficient Jurkat cells are resistant to mitochondrial apoptosis. (a) Expression of p56Lck was analysed in parental Jurkat cells, Jurkat cells overexpressing Bcl-2, Lck-deficient JCaM1.6 cells and their Lck-transfected derivatives by Western blot analysis with an antibody against the C-terminus of Lck. (b) Apoptosis induction by chemotherapeutic drugs. Parental Jurkat cells, JCaM and JCaM/Lck cells were either left untreated or incubated with etoposide (5 μg/ml), daunorubicin (1 μg/ml), doxorubicin (1 μg/ml) or mitomycin C (5 μg/ml). Apoptosis was assessed after the indicated times by propidium iodide staining of hypodiploid nuclei. (c and d) Effect of Lck deficiency on CD95 death receptor-mediated cell death. (c) JCaM and JCaM/Lck cells were treated for the indicated time with anti-CD95 moAb (1 μg/ml) or staurosporine (2.5 μ M), before apoptosis was determined by measurement of hypodiploid DNA. (d) Comparison of CD95- and staurosporine-mediated apoptosis in Jurkat wild-type, Jurkat-Bcl-2, JCaM and JCaM/Lck cells after 24 h of incubation with anti-CD95 or staurosporine. The data show that early apoptosis is slightly attenuated in JCaM cells, whereas Lck deficiency does not protect against CD95 at later time points.

JCaM cells were also resistant to apoptosis induced by staurosporine, a classical inducer of the mitochondrial apoptosis pathway, and again re-expression of Lck sensitized the cells to apoptosis (Figure 1c and d). The resistance of JCaM cells was in a similar range as in Jurkat cells overexpressing Bcl-2. However, regardless of the Lck status, both Lck-deficient and -proficient cells were almost equally sensitive to death receptor CD95 ligation when measured after 24 h (Figure 1d); a slight protection was only seen at early time points (Figure 1c). Similarly, CD95-mediated apoptosis was only delayed but not prevented in Bcl-2-overexpressing Jurkat cells. The selective protection contributed by Lck deficiency against drug-triggered, but not CD95-mediated apoptosis therefore indicated that Lck interfered with the mitochondrial pathway of apoptosis.

Lck inhibits the mitochondrial caspase cascade

The mitochondrial pathway is triggered by the release of cytochrome c, which together with Apaf-1 binds to procaspase-9 to form the apoptosome and results in the activation of effector caspase-3, -6 and -7. To determine in more detail the reason for the inability of Lck-deficient cells to undergo apoptosis, we monitored the release of cytochrome c from mitochondria and the activation of caspase-3 and -9. In Lck-expressing cells, but not in Lck-deficient cells, etoposide treatment resulted in the marked activation of caspase-3 within 3–4 h, as determined by the proteolytic processing from its 32 kDa precursor into the 17 kDa active subunit (Figure 2a). A similar result was obtained when activation of caspase-3 was measured by fluorogenic substrate assays or by the cleavage of its substrate PARP-1 (data not shown). The cleavage of caspase-3 was preceded by the activation of caspase-9 that was only observed in Lck-expressing but not in Lck-deficient JCaM cells (Figure 2a). Almost coincident with the activation of caspase-9 was the mitochondrial release of cytochrome c, which was exclusively seen in the presence of Lck.

Figure 2
figure2

Cytochrome c release and caspase activation in chemotherapeutic drug-induced apoptosis depends on Lck. (a) JCaM and JCaM/Lck cells were treated for the indicated time with etoposide (5 μg/ml). (a) Cellular proteins were separated by SDS–PAGE, and the proteolytic processing of caspase-9 (upper panel) and caspase-3 (middle panel) was detected by immunoblotting. Open arrowheads indicate the uncleaved and closed arrowheads the cleaved forms of the indicated proteins. For measurement of cytochrome c release (lower panel), cells were homogenized and the S10 fraction depleted of mitochondria was analysed by SDS–PAGE. An unspecific protein band that served as a control for equal protein loading is indicated with an asterisk. (b) Proteolytic processing of caspase-8 and Bid was analysed after the indicated times of etoposide treatment.

The mitochondrial pathway is connected with the death receptor pathway by the Bcl-2 protein Bid that, upon cleavage by caspase-8, translocates to the mitochondria to trigger the release of cytochrome c. In JCaM/Lck but not in JCaM cells, the truncated p15 form of Bid began to appear within 4 h after etoposide treatment (Figure 2b). In addition, cleavage of caspase-8 into the p18 subunit was only observed in Lck-expressing cells. In comparison to cytochrome c release and caspase-9 processing, the cleavage of Bid and caspase-8 was delayed, indicating that it occurred by a postmitochondrial event, as also suggested by others (Engels et al., 2000; Tang et al., 2000; von Haefen et al., 2003; Sohn et al., 2005).

The proapoptotic effect of Lck is not mediated by Akt and MAP kinases

It has been demonstrated that MAP kinase and Akt pathways can interfere with mitochondrial apoptosis. Akt provides a survival signal by inactivating proapoptotic molecules such as Bad and Forkhead transcription factor FKHRL1 (Datta et al., 1999). MAP kinase cascades can target different Bcl-2 proteins and in most cases promote their apoptotic activity. Since activation of these mediators has been reported to occur following stimulation of Lck, we investigated their potential involvement in Lck-mediated apoptosis. As determined with phosphospecific antibodies, p38 was activated within 3–4 h of etoposide incubation in Lck-expressing JCaM cells but not in the Lck-deficient counterparts (Figure 3a). Within a similar time course, activation of Erk1/2 was observed only in the presence of Lck. JNK activation was detected slightly earlier and was only seen in the Lck-expressing cells. The constitutive expression of either kinase did not differ in the two cell lines. Importantly, however, UO126, an inhibitor of the Erk pathway, and SP600125, a JNK inhibitor, did not interfere with apoptosis induction (Figure 3b). Inhibition of p38 by SB203580 had also no effect (data not shown), suggesting that MAP kinase cascades were at least not directly involved in apoptosis induction.

Figure 3
figure3

Protection against apoptosis in Lck-deficient cells does not involve MAP kinases, Akt or c-Myc. (a) Time course of kinase activation and c-Myc expression in JCaM and JCaM/Lck cells during etoposide-induced apoptosis. Cells were incubated for the indicated times with 5 μg/ml etoposide, before total cell extracts were investigated for the expression of p38, Erk1/2, JNK, Akt and c-Myc by immunoblot analysis. Activation of the kinases was measured using phosphospecific antibodies. (b) Effect of kinase inhibitors on etoposide-induced apoptosis in JCaM/Lck cells. Cells were either left untreated (control) or pretreated with the MEK inhibitor U0126 or the JNK inhibitor SP600125, and then incubated with etoposide. Apoptosis was assessed after 12 h; the results show means of two independent experiments measured in triplicate.

Treatment of both cell lines with etoposide also did not affect the activation of Akt (Figure 3a). Lck-expressing cells even showed a slightly stronger phosphorylation of Akt. Moreover, no differences were found in the expression levels of c-Myc. Using gel retardation analysis, we also investigated the activation of transcription factors AP-1, NF-AT and NF-κB, which might provide a T-cell survival signal. In contrast to classical proinflammatory stimuli, etoposide treatment did not induce a significant activation of the transcription factors nor were there differences between both cell lines (data not shown). Thus, these results demonstrate that neither the activation of antiapoptotic transcription factors or Akt nor effects mediated by MAP kinases were involved in the apoptosis resistance mediated by Lck deficiency.

Lck specifically controls Bak expression

The mitochondrial pathway is controlled by the ratio of pro- and antiapoptotic Bcl-2 proteins. Since Lck deficiency resulted in an apoptosis defect probably upstream of cytochrome c release, we investigated the expression of various proapoptotic Bcl-2 family members, such as Bax, Bak and Bad, and anti-apoptototic proteins, such as Bcl-2 and Bcl-xL. Unexpectedly, apoptosis-resistant JCaM cells revealed even slightly reduced levels of Bcl-2 and Bcl-xL compared to parental Jurkat and Lck-transfected JCaM cells. This might concur with the observation that Lck stimulates the expression of these antiapoptotic molecules. Jurkat cells and their Lck-deficient counterparts expressed similar levels of Bax. Striking, however, was our observation that expression of the Bax-related proapoptotic Bcl-2 member Bak was almost completely absent in JCaM cells, whereas re-expression of Lck resulted in expression levels of Bak similar to wild-type Jurkat cells (Figure 4a).

Figure 4
figure4

Expression status of Bcl-2, IAP family proteins and NF-κB RelA in the different Jurkat cell lines. (a) Total cell extracts of JCaM, JCaM/Lck, parental and Bcl-2-overexpressing Jurkat cells were immunoblotted against the antiapoptotic proteins Bcl-xL and Bcl-2 as well as against the proapoptotic family members Bad, Bax and Bak (left panels). Cell extracts were analysed in parallel for the expression of the IAP family members c-IAP1, c-IAP2 and XIAP as well as RelA (right panels). Immunoblotting against actin served as a control for equal protein loading. (b) Subcellular localization of cytochrome c and Bcl-2 proteins during etoposide-induced apoptosis. JCaM/Lck and JCaM cells were treated for the indicated times with etoposide and fractionated in a cytosolic and a heavy-membrane fraction containing mitochondria. The fractions were immunoblotted against different Bcl-2 proteins. Actin served as control for equal protein loading of cytosolic extracts. Fractions were reprobed with an antibody against the outer mitochondrial membrane marker Tom20. Note that expression of Bak is completely absent in JCaM cells.

We also compared the expression levels of inhibitor of apoptosis proteins (IAPs), which directly bind to and inhibit active caspases. However, expression of neither c-IAP1 and c-IAP2 nor of XIAP differed in the individual cell lines (Figure 4a), indicating that these apoptosis inhibitors were presumably not responsible for the apoptosis-resistant phenotype of Lck-deficient cells.

We next investigated the subcellular localization of the Bcl-2 proteins in the course of etoposide treatment and therefore prepared cytosolic and mitochondrial fractions of JCaM cells and their Lck-expressing counterparts. In support of the previous result, cytochrome c was only released from mitochondria of Lck-expressing but not of Lck-deficient cells. Both Bcl-2 and Bcl-xL were mainly detected in the mitochondrial fraction and remained relatively constant during etoposide treatment (Figure 4b). Also, Bax was mainly found at the mitochondria. Importantly, fractionation in cytosolic and mitochondrial extracts confirmed that Bak was strikingly absent in JCaM cells, whereas considerable levels were detected in mitochondria from JCaM/Lck cells. In line with the previous data, during drug treatment Bak was not translocated from the cytosol but constitutively found in the mitochondrial compartment.

To investigate whether the differential expression of Bak was also detectable at the mRNA level, we performed reverse transcriptase–polymerase chain reaction (RT–PCR) analyses. As shown in Figure 5a, in JCaM cells Bak-specific transcripts were almost completely absent, whereas Bak mRNA was expressed in the parental Jurkat and Lck-transfected cells. This expression pattern did not change when JCaM and JCaM/Lck cells were stimulated with etoposide or phytohemagglutinin (Figure 5b). Stimulation of the cells also did not affect the expression levels of other pro- and antiapoptotic Bcl-2 proteins. Together, these data show that Lck strongly regulates Bak expression at the transcriptional level.

Figure 5
figure5

Transcriptional expression of Bcl-2 family members in Lck-deficient and -proficient Jurkat cells. (a) RNA from JCaM, JCaM/Lck and parental Jurkat cells was subjected to RT–PCR using oligonucleotide primers specific for Bax, Bak, Bcl-2 and Bcl-x. The PCR products were separated on agarose gels and visualized by ethidium bromide staining. The closed arrowheads indicate the relevant PCR products and the open arrowheads a GAPDH PCR product that was amplified in parallel. (b) Expression of Bcl-2 family members in JCaM/Lck and JCaM cells during phytohemagglutinin (PHA) and etoposide treatment. Cells were treated for the indicated time with PHA (50 μg/ml) or etoposide (5 μg/ml), before RNA was extracted and subjected to RT-PCR analysis.

Bak undergoes a proapoptotic conformational change in Lck-expressing cells and mediates cytochrome c release both in vitro and in vivo

Recent data show that mitochondrial apoptosis is regulated by the multidomain Bcl-2 proteins Bax or Bak, which undergo a N-terminal conformational change resulting in cytochrome c release. The conformational change of Bak and Bax can be analysed using specific antibodies against their normally occluded N-terminus. FACS analysis of JCaM cells with such antibodies revealed only a slight activation of Bax after etoposide treatment, whereas due to its absence, no conformational change of Bak was observed (Figure 6a). In contrast, JCaM/Lck cells showed a pronounced conformation change of Bak upon apoptosis induction (Figure 6b). Interestingly, however, under these conditions JCaM/Lck cells also displayed a stronger activation of Bax compared to the Bak-deficient JCaM cells. This result therefore suggests a degree of functional cooperation of Bak and Bax regarding their conformational activation.

Figure 6
figure6

Requirement of Lck for Bak and Bax conformational change. JCaM (a) and JCaM/Lck cells (b) were either left untreated or incubated for 4 h with etoposide. The conformational change was measured by flow cytometry using activation-specific antibodies against Bax and Bak. The filled histograms show the staining with isotype-matched control antibodies and the grey line indicates the specific staining for the active forms of Bak and Bax.

To further substantiate these findings, we treated mitochondria in digitonin-permeabilized JCaM/Lck cells with recombinant tBid, representing the proapoptotic, caspase-8 generated form of Bid. The addition of exogenous tBid rapidly induced the conformational change of Bak in vitro (Figure 7a). The effect was associated with the release of cytochrome c into the supernatant and a corresponding reduction of cytochrome c in mitochondria (Figure 7b). The translocation of cytochrome c by tBid was specifically observed in JCaM/Lck cells, but virtually absent in mitochondria from Lck-deficient cells. These in vitro data therefore confirm that the defect of the apoptosis-resistant and Lck-deficient cells is directly located at the mitochondria and mediated by Bak deficiency.

Figure 7
figure7

Induction of conformational Bak activation and cytochrome c release by tBid in vitro. (a) JCaM/Lck cells were permeabilized and then either left untreated or treated with 2 μ M recombinant tBid. After 30 min, the conformational change of Bak and Bax was analysed as described in Figure 6. (b) tBid induces cytochrome c release from mitochondria of JCaM/Lck but not of JCaM cells. Isolated mitochondria from both cells were treated with 2 μ M tBid for 30 min. Cytochrome c was then measured in the supernatant and mitochondrial pellet by immunoblot analysis. The blots were reprobed with anti-Bak confirming the absence of Bak in mitochondria from JCaM cells.

T-cell signaling and kinase activity of Lck are not required for apoptosis

It was reported that some biological effects of Lck require different structural domains (Denny et al., 1999). We therefore investigated the specificity of the proapoptotic effect of Lck and its downstream signaling. Treatment of JCaM/Lck cells with etoposide resulted in the activation of Lck as measured by its autophosphorylation (Figure 8a). In comparison to TCR stimulation by anti-CD3, the activation of Lck by etoposide was slightly delayed but clearly visible. Furthermore, treatment of cells with the Src kinase inhibitor herbimycin A, which targets Lck for proteasomal degradation (Sepp-Lorenzino et al., 1995), strongly prevented apoptosis induction by etoposide (Figure 8b). Surprisingly, however, the pyrazolopyrimidine compound PP2, which is a direct inhibitor of the kinase activity of Lck (Hanke et al., 1996), was completely ineffective in inhibiting cell death, even when high concentrations were used (Figure 8b). This result therefore indicates that the kinase activity is not required for the proapoptotic effect of Lck.

Figure 8
figure8

Role of Lck kinase activity and downstream T-cell mediators in etoposide-induced apoptosis. (a) Induction of Lck kinase activity. JCaM and JCaM/Lck cells were stimulated for the indicated times with etoposide or anti-CD3. Lck activity was measured in an autophosphorylation assay. (b) Effect of kinase inhibitors. JCaM and JCaM/Lck cells were either left untreated or pretreated with 10 μ M herbimycin A or 20 μ M PP2, before apoptosis was induced by etoposide. After 24 h, the number of apoptotic cells was measured by their DNA content and is indicated as mean value±s.d. of two independent experiments performed in triplicate. (c) Comparison of apoptosis in individual Jurkat cell lines deficient in different T-cell signaling mediators. Parental Jurkat cells, Lck-deficient and -proficient JCaM cells, Jurkat cells deficient for LAT or ZAP-70 as well as JCaM cells transfected with a myristoylation-deficient Lck construct were either left untreated or stimulated for 24 h with etoposide, before apoptosis was assessed by measurement of hypodiploid DNA. The results show the mean±s.d. of three experiments measured in triplicate. (d) Bak expression in the different T-cell clones. The status of Bak was investigated in the cells described in (c) by Western blot analysis. An unspecific protein band that served as a control for equal protein loading is indicated with an asterisk.

During T-cell stimulation, Lck phosphorylates several signaling mediators, most importantly ZAP-70, which then phosphorylates key adapter proteins, including LAT (linker for activation of T cells). Loss of ZAP-70 or LAT disrupts TCR signaling and blocks T-cell activation (Finco et al., 1998; Williams et al., 1998). To analyse whether these key mediators of T-cell signaling were involved in the proapoptotic effect of Lck, we used ZAP-70- and LAT-deficient Jurkat cells (Figure 8c). However, cells deficient for LAT underwent apoptosis to a similar extent as their LAT-transfected counterparts or JCaM/Lck cells, and neither was apoptosis sensitivity impaired in ZAP-70-deficient Jurkat cells. Moreover, apoptosis was also not blocked in JCaM cells retransfected with a myristoylation-deficient Lck construct, indicating that Lck myristoylation, which is essential for T-cell stimulation, was not required for apoptosis sensitization. In addition, neither cells expressing the myristoylation-deficient Lck nor cells deficient for ZAP-70 or LAT showed defects in Bak expression (Figure 8d), suggesting that different signals are required for T-cell stimulation and the apoptosis-sensitizing effect of Lck.

Discussion

The present study demonstrates a critical role of Lck for induction of apoptosis in human leukemic cells in response to anticancer drugs. The deficiency in Lck endowed Jurkat cells with a potent resistance mechanism to various stimuli that activate the mitochondrial pathway, whereas it did not block apoptosis triggered by death receptors. Moreover, apoptosis resistance was completely reversed in cells with reconstituted Lck expression. We also demonstrate that the apoptosis-resistant phenotype of Lck-deficient cells is caused by the selective defect of Bak expression, suggesting that Lck is not only a mediator of chemosensitivity but also a hitherto unknown regulator of Bak expression.

At first view, it appears surprising that Lck which has been mainly linked to T-cell activation regulates apoptosis. However, the proliferation of cells requires not only mitogenic signals but also survival signals, which provide a safeguard against deregulated proliferation. Thus, mitogenic signaling without accompanying survival signals triggers apoptosis and leads to the elimination of potentially malignant cells. The oncogenes c-Myc and E2F, for example, which generate strong proliferative signals, induce apoptosis in the absence of exogenous survival factors (Green and Evan, 2002). Nonreceptor tyrosine kinases of the Src family might also exert such a dual role in proliferation and apoptosis. Lck appears to be required for ceramide-, drug- and irradiation-induced apoptosis (Belka et al., 1999; Manna et al., 2000; Gruber et al., 2004), while the B-cell receptor-associated Src-like kinases Btk and Lyn have been implicated in apoptosis in response to radiation (Uckun et al., 1996; Qin et al., 1997; Hernandez-Hansen et al., 2004). Interestingly, peripheral T cells from mice with inducible Lck deficiency were reported to have prolonged survival in vivo, although the role of altered apoptosis has not been addressed in this study (Seddon et al., 2000). Also c-Src and v-Src can sensitize cells to apoptosis (Webb et al., 2000; Melendez et al., 2004). Recently, it has been demonstrated that overexpression of two Drosophila Src kinases not only induces ectopic cell proliferation but also apoptosis in transgenic flies (Pedraza et al., 2004). Src-induced proliferation and apoptosis were found to be largely independent events, as blocking ectopic proliferation did not block cell death in Drosophila.

Our study demonstrates that the apoptotic role of Lck is also independent of its principal function in T-cell signaling as well as its kinase activity. Following TCR engagement, Lck mediates phosphorylation ZAP-70 and LAT, and ultimately promotes the activation of downstream pathways. Using Jurkat clones deficient in these T-cell mediators, we clearly show that ZAP-70 and LAT are not involved in Lck-mediated apoptosis resistance. Moreover, myristoylation, which is essential for targeting Lck to the plasma membrane during T-cell stimulation, was not required, as JCaM cells reconstituted with a myristoylation-deficient Lck mutant were equally sensitive to apoptosis as parental Jurkat cells or cells reconstituted with wild-type Lck.

To investigate the mechanism of chemoresistance in Lck-deficient cells, we investigated several pathways involved in apoptosis regulation. Inhibitors of stress-responsive kinases including Erk, p38 and JNK did not affect apoptosis sensitivity of Jurkat cells in response to various chemotherapeutic drugs. Furthermore, no differences in the activation profile of Akt or the transcription factors NF-AT, AP-1 and NF-κB were observed between Lck-deficient and -proficient cells, and neither were the expression levels of various IAP members altered, suggesting that none of these classical apoptosis pathways was involved in the resistance caused by the lack of Lck.

We found that the block in the apoptotic cascade in Lck-deficient cells was rather localized to the mitochondrial apoptosis machinery. No cytochrome c release or caspase activation was detected in Lck-deficient cells treated with chemotherapeutic drugs. Furthermore, in contrast to mitochondria from wild-type cells or cells reconstituted with Lck, those from Lck-deficient cells did not release cytochrome c in response to tBid in vitro. This observation indicates that the apoptotic defect of Lck-deficient cells was directly localized at mitochondria. Investigating the expression of Bcl-2 family members, we found basically no differences in the expression levels of several anti- or proapototic proteins. However, Bak, which in addition to Bax is considered as the gatekeeper of cytochrome c release, was conspicuously absent in Lck-deficient cells. Importantly, reconstitution of Lck fully restored Bak expression to a similar level as in wild-type cells.

The critical role of Bak in Lck-mediated apoptosis sensitization was substantiated by experiments with conformation-specific antibodies that allow the measurement of Bak and Bax activation. Interestingly, after treatment of JCaM cells with genotoxic agents, a weak conformational change of Bax occurred, whereas JCaM/Lck cells displayed a stronger activation of Bax compared to the Bak-deficient JCaM cells. As previously reported (Mikhailov et al., 2003; Lindenboim et al., 2005), this might indicate a functional cooperation between Bak and Bax with regard to their conformational activation. It has been proposed that tBid acts partly by inducing conformational changes in Bax (Eskes et al., 2000). However, tBid can also trigger cytochrome c release also independently of Bax (Desagher et al., 1999). Furthermore, Bak-deficient hepatocytes and T cells are resistant to tBid induction of cytochrome c release (Wei et al., 2000; Wang et al., 2001). This therefore suggests that at least in certain cell types, Bak rather than Bax is the key regulator of mitochondrial apoptosis and might determine the chemoresistance of tumor cells.

Bak and Bax are generally assumed to substitute for each other, since deficiency for both genes is required to render cells resistant to a number of proapoptotic agents, while single knockouts for either gene have far less effects. Bak and Bax single knockout mice are rather normal, whereas most of the mice lacking both genes display multiple developmental defects (Lindsten et al., 2000), suggesting that Bax and Bak have overlapping roles in the regulation of apoptosis. These overlapping roles, which were observed in murine models for development of normal cells, remain to be elucidated in human tumor cells. Despite their regulation by p53, Bak and Bax also have proapoptotic effects in cells with altered p53 expression (Knudson et al., 2001; Degenhardt et al., 2002). Therefore, a redundant function for Bax and Bak, as detected in normal murine cells, may be differentially regulated in cancer cells with mutated p53, such as human leukemic cells. It is possible that in tumor cells the balance between Bax and Bak levels of expression is different from that of normal cells. Moreover, although Bak and Bax might have overlapping functions, it has been shown that they may serve different roles depending on the proapoptotic stimulus (Panaretakis et al., 2002; Cartron et al., 2003). For instance, induction of cell death by the BH3-only protein Nbk/Bik is entirely Bax dependent, but does not require Bak (Gillissen et al., 2003). It is also conceivable that Bak and Bax could be sequestered by different antiapoptotic Bcl-2 members that are targeted by distinct BH3 proteins (Willis et al., 2005).

Lck deficiency resulted in a lack of Bak expression at the mRNA level, suggesting Lck regulates transcriptional activation of the Bak gene. So far, transcriptional regulation of most Bcl-2 proteins is poorly understood. Various proapoptotic Bcl-2 members including Bax, Puma, Noxa and Bid are p53 target genes in the DNA damage response pathway. However, Jurkat cells lack functional p53 (Cheng and Haas, 1990; Iwamoto et al., 1996), indicating that Lck-mediated regulation of Bak expression is p53 independent. In contrast to Bax, the transcriptional regulation of Bak has not yet been analysed at the promoter level. Computer analysis of its 3.5 kb upstream region of the Bak gene reveals no TATA-box, but putative binding sites for NF-κB, SP1 and p53 (data not shown). Moreover, an ISRE and GAS site is present that could bind IRF and STAT family members. The latter sites are particularly interesting, as they might be at least partially responsible for the apoptosis resistance conferred by targeted gene deletion of certain IRF and STAT family members (Tamura et al., 1995; Battle and Frank, 2002).

In summary, our data indicate a hitherto unrecognized link between the tyrosine kinase Lck, Bak expression and chemoresistance. Although mutations in the Bak gene were detected in human gastric and colorectal cancers (Kondo et al., 2000), Bak deficiency as a mechanism for chemoresistance has not yet been addressed. The resistance to anticancer drugs suggests that the deficiency of Lck and a consequent failure to express Bak participates in the regulation of mitochondrial apoptosis in leukemic cancer cells.

Materials and methods

Cell lines and reagents

All cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 100 U of penicillin/ml and 0.1 mg streptomycin/ml (PAA Laboratories, Linz, Austria). The Lck-deficient Jurkat cell clone JCaM1.6 (Straus and Weiss, 1992) and Lck-retransfected JCaM1.6 cells as well as JCaM2 cells deficient for LAT (Finco et al., 1998) and its retransfected derivatives were kindly provided by A Weiss (San Francisco, USA). Jurkat cells deficient for ZAP-70 (Williams et al., 1998) were a gift from RT Abraham (Durham, NC, USA). JCaM1.6 cells stably transfected with a myristoylation-deficient Lck construct were obtained from A Kosugi (Osaka, Japan). Jurkat cells overexpressing Bcl-2 have been described previously (Stepczynska et al., 2001). Staurosporine, etoposide, doxorubicin, daunorubicin, mitomycin C and propidium iodide were purchased from Sigma (Deisenhofen, Germany). Agonistic anti-CD95 and caspase-8 moAb were from BioCheck (Münster, Germany). Anti-Bid, anti-caspase-3 moAb and polyclonal anti-caspase-9 were from R&D Systems (Wiesbaden, Germany) and New England BioLabs (Beverly, MA, USA), respectively. Anti-Bax and cytochrome c moAbs were from PharMingen (Hamburg, Germany). The conformation-specific antibodies rabbit anti-Bax-NT and monoclonal anti-Bak were from Upstate Biotechnology (Lake Placid, NY, USA) and Oncogene, respectively. MoAbs against Lck, actin, Bcl-2 and c-Myc, and antisera against c-IAP1 and c-IAP2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Tom20 MoAb and polyclonal anti-Bcl-xL were from Transduction Laboratory (Heidelberg, Germany). Anti-JNK, anti-p38 MAPK, anti-p44/42 (Erk-1), and anti-Akt were obtained from Cell Signaling. Activation of the kinases was monitored with anti-phospho-p38 (T180/Y182) and anti-phospho-Akt (Y326) rabbit antisera as well as with mouse anti-phospho-SAPK/JNK (T183/Y185) antibody. Anti-phospho-p44/42 Erk (T202/Y204) moAbs were obtained from Cell Signaling.

Treatment of cells and measurement of cell death

Cells were exposed to anti-CD95 (1 μg/ml) or the drugs staurosporine (2.5 μ M), etoposide (5 μg/ml), doxorubicin (1 μg/ml), daunorubicin (1 μg/ml) and mitomycin C (5 μg/ml). Inhibitors obtained from Calbiochem were used at the following concentrations: SP600125 (1 μ M), UO126 (10 μ M), PP2 (20 μ M) and herbimycin A (10 μ M). At the concentrations used, the inhibitors did not elicit any cytotoxic response on their own. To determine the level of apoptosis, 3 × 104 cells/well were seeded in microtiter plates and treated for the indicated time with anti-CD95 or the chemotherapeutic agents. The leakage of fragmented DNA from apoptotic nuclei was measured as described (Essmann et al., 2003). Briefly, apoptotic nuclei were prepared in a hypotonic lysis buffer (1% sodium citrate, 0.1% Triton X-100, 50 μg/ml propidium iodide) and analysed by flow cytometry. Nuclei to the left of the 2N peak containing hypodiploid DNA were considered as apoptotic. All flow cytometric analyses were performed on a FACScalibur (Becton Dickinson).

Flow cytometric analysis of Bak and Bax conformational changes

At the indicated times after drug treatment, cells (1 × 106) were harvested by centrifugation at 300 g for 5 min, washed with PBS and fixed in PBS/0.5% paraformaldehyde on ice for 30 min. Cells were then washed three times in PBS/1% FCS. Staining with conformation-specific antibodies against Bax and Bak and isotype-matched control antibodies was performed with an 1:50 dilution of the respective antibody in 50 μl staining buffer (PBS, 1% FCS, 50 μg/ml digitonin). Then, cells were washed three times and resuspended in 50 μl staining buffer containing 0.1 μg Alexafluor 488-labeled chicken anti-mouse or FITC-coupled goat anti-rabbit IgG (Molecular Probes) and incubated on ice for 30 min in the dark. After three washing steps, conformational changes of Bak and Bax were immediately measured in the FL-1 channel of a flow cytometer.

Preparation of cytosolic, mitochondrial and total cell extracts

After drug treatment, cells were washed in PBS and suspended in low-salt lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonyl fluoride (PMSF) and 3 μg/ml of each aprotinin, leupeptin and pepstatin A. After 20 min on ice, cells were lysed by passing them three times through a 26 G needle. Lysates were then centrifuged at 500 g for 10 min to remove nuclei and intact cells. The supernatants were centrifuged at 10 000 g for 30 min at 4°C to obtain a cytosolic fraction. The resulting pellet that contained crude mitochondria was lysed in high-salt lysis buffer (1% NP-40, 20 mM HEPES, pH 7.9, 2 mM PMSF, 350 mM NaCl, 20% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT and protease inhibitors. For total cell extracts, cells were directly lysed in a high-salt buffer and centrifuged at 10 000 g for 30 min.

Mitochondria isolation

To obtain mitochondria for in vitro cytochrome c release assays, cells were permeabilized in buffer A containing 50 μg/ml digitonin, 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4, 1 mM DTT and protease inhibitors, and immediately passed three times through a 26 G needle. After centrifugation at 500 g for 10 min, the remaining supernatants were further centrifuged at 10 000 g for 30 min at 4°C. The resulting crude mitochondrial pellets were washed three times with buffer B containing 250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4, to remove microsomal contaminations. The crude mitochondrial fraction was then resuspended in 10 mM HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 4.2 mM sodium succinate, 1 mM DTT and protease inhibitors, and used for analysis of cytochrome c release (Bantel et al., 2001).

Purification of recombinant tBid and in vitro cytochrome c release assay

The caspase-8-generated fragment of Bid was cloned in PGEX-2T, a bacterial expression vector for glutathione S-transferase fusion proteins. After induction of protein expression, tBid was purified by glutathione sepharose 4B beads (Amersham Bioscience, Uppsala, Sweden). The purified fractions were dialysed in PBS and examined for homogeneity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Purified mitochondria from JCaM and JCaM/Lck cells were incubated with GST-tBid at 37°C for 30 min. Then, the samples were centrifuged at 10 000 g for 20 min. The resulting supernatant and mitochondrial pellets were analysed by immunoblotting.

Immunoblotting

Proteins (30 μg/lane) were separated under reducing conditions on 10–15% SDS–PAGE and electroblotted to a polyvinylidene difluoride membrane. Membranes were blocked for 2 h with 5% milk powder in Tris-buffered saline, and then incubated overnight at 4°C with the primary antibodies as recommended by the manufacturers. Membranes were washed four times with TBS/0.02% Triton X-100 and incubated with the respective peroxidase-conjugated secondary antibody for 1 h. Following extensive washing, the reaction was developed by enhanced chemiluminescent staining.

Reverse transcriptase–polymerase chain reaction

The mRNA expression levels of the different Bcl-2 members were monitored by RT–PCR. Total RNA was extracted from the cells with TRI reagent (Sigma). RT and PCR were performed using the Titanium One-step RT–PCR kit (BD Biosciences). For standardization, each RT sample was subjected to PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The 3′ and 5′ primers used for amplification were 5′-IndexTermATG TCT CAG AGC AACCCG GAG-3′ and 5′-IndexTermTTT CCG ACT GAA GAG TGA GCC C-3′ for Bcl-x, 5′-IndexTermGAA GAT CTG CTT CGG GGC AAG GCC CAG GT-3′ and 5′-IndexTermAAG GAT CCT CAT GAT TTG AAG AAT CTT CG-3′ for Bak, 5′-IndexTermGAA GAT CTG ACG GGT CCG GGG AGC AGC CC-3′ and 5′-IndexTermAAG GAT CCT CAG CCC ATC TTC TTC CAG AT-3′ for Bax, 5′-IndexTermATG GCG CACGCT GGG AGA AC-3′ and 5′-IndexTermCCA GCC TCC GTT ATC CTG GATC-3′ for Bcl-2, and 5′-IndexTermGTG GAA GGA CTC ATG ACC ACA G-3′ and 5′-IndexTermCTG GTG CTC AGT GTA GCC CAG-3′ for GAPDH.

Lck kinase assay

Cells were treated for the indicated time with etoposide or anti-CD3 (OKT3), and then extracted in a buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF and 1 mM DTT. Cell extracts were immunoprecipitated with 1 μg anti-Lck for 12 h at 4°C followed by incubation with protein A sepharose beads for 1 h. The beads were washed with lysis buffer and kinase buffer (50 mM HEPES, pH 7.4, 20 mM NaCl, 10 mM MgCl2, 100 μ M Na3VO4, 500 μ M DTT, 25 mM β-glycophosphate), and then assayed in kinase buffer containing 10 μCi γ[32P]ATP and 2.5 μ M ATP at 37°C for the indicated times. Reactions were stopped by the addition of sample buffer and subjected to SDS–PAGE.

References

  1. Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I et al. (1997). Science 18: 370–372.

  2. Bantel H, Sinha B, Domschke W, Peters G, Schulze-Osthoff K, Janicke RU . (2001). J Cell Biol 155: 637–648.

  3. Battle TE, Frank DA . (2002). Curr Mol Med 2: 381–392.

  4. Belka C, Marini P, Lepple-Wienhues A, Budach W, Jekle A, Los M et al. (1999). Oncogene 18: 4983–4992.

  5. Cartron PF, Juin P, Oliver L, Martin S, Meflah K, Vallette FM . (2003). Mol Cell Biol 23: 4701–4712.

  6. Cheng J, Haas M . (1990). Mol Cell Biol 10: 5502–5509.

  7. Cory S, Huang DC, Adams JM . (2003). Oncogene 22: 8590–8607.

  8. Daniel PT, Schulze-Osthoff K, Belka C, Guner D . (2003). Essays Biochem 39: 73–88.

  9. Datta SR, Brunet A, Greenberg ME . (1999). Genes Dev 13: 2905–2927.

  10. Degenhardt K, Chen G, Lindsten T, White E . (2002). Cancer Cell 2: 193–203.

  11. Denny MF, Kaufman HC, Chan AC, Straus DB . (1999). J Biol Chem 274: 5146–5152.

  12. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S et al. (1999). J Cell Biol 144: 891–901.

  13. Engels IH, Stepczynska A, Stroh C, Lauber K, Berg C, Schwenzer R et al. (2000). Oncogene 19: 4563–4573.

  14. Eskes R, Desagher S, Antonsson B, Martinou JC . (2000). Mol Cell Biol 20: 929–935.

  15. Essmann F, Bantel H, Totzke G, Engels IH, Sinha B, Schulze-Osthoff K et al. (2003). Cell Death Differ 10: 1260–1272.

  16. Ferri KF, Kroemer G . (2001). Nat Cell Biol 3: E255–E263.

  17. Finco TS, Kadlecek T, Zhang W, Samelson LE, Weiss A . (1998). Immunity 9: 617–626.

  18. Fischer U, Janicke RU, Schulze-Osthoff K . (2003). Cell Death Differ 10: 76–100.

  19. Gillissen B, Essmann F, Graupner V, Starck L, Radetzki S, Dorken B et al. (2003). EMBO J 22: 3580–3590.

  20. Gonzalez-Garcia A, Borlado LR, Leonardo E, Merida I, Martinez A, Carrera AC . (1997). J Immunol 158: 4104–4112.

  21. Green DR, Evan GI . (2002). Cancer Cell 1: 19–30.

  22. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM et al. (1999). J Cell Biol 144: 903–914.

  23. Gruber C, Henkel M, Budach W, Belka C, Jendrossek V . (2004). Biochem Pharmacol 67: 1859–1872.

  24. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ et al. (1996). J Biol Chem 271: 695–701.

  25. Hernandez-Hansen V, Mackay GA, Lowell CA, Wilson BS, Oliver JM . (2004). J Leukocyte Biol 75: 143–151.

  26. Hur YG, Yun Y, Won J . (2004). J Immunol 172: 79–87.

  27. Iwamoto KS, Mizuno T, Ito T, Tsuyama N, Kyoizumi S, Seyama T . (1996). Cancer Res 56: 3862–3865.

  28. Kane LP, Lin J, Weiss A . (2000). Curr Opin Immunol 12: 242–249.

  29. Klee M, Pimentel-Muinos FX . (2005). J Cell Biol 168: 723–734.

  30. Knudson CM, Johnson GM, Lin Y, Korsmeyer SJ . (2001). Cancer Res 61: 659–665.

  31. Kondo S, Shinomura Y, Miyazaki Y, Kiyohara T, Tsutsui S, Kitamura S et al. (2000). Cancer Res 60: 4328–4330.

  32. Lindenboim L, Kringel S, Braun T, Borner C, Stein R . (2005). Cell Death Differ 12: 713–723.

  33. Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA et al. (2000). Mol Cell 6: 1389–1399.

  34. Manna SK, Aggarwal BB . (2000). J Immunol 164: 5156–5166.

  35. Manna SK, Sah NK, Aggarwal BB . (2000). J Biol Chem 275: 13297–13306.

  36. Melendez J, Turner C, Avraham H, Steinberg SF, Schaefer E, Sussman MA . (2004). J Biol Chem 279: 53516–53523.

  37. Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White E, Saikumar P . (2003). J Biol Chem 278: 5367–5376.

  38. Panaretakis T, Pokrovskaja K, Shoshan MC, Grander D . (2002). J Biol Chem 277: 44317–44326.

  39. Pedraza LG, Stewart RA, Li DM, Xu T . (2004). Oncogene 23: 4754–4762.

  40. Puthalakath H, Strasser A . (2002). Cell Death Differ 9: 505–512.

  41. Qin S, Minami Y, Kurosaki T, Yamamura H . (1997). J Biol Chem 272: 17994–17999.

  42. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME . (1998). Eur J Biochem 254: 439–459.

  43. Schwerk C, Schulze-Osthoff K . (2003). Biochem Pharmacol 66: 1453–1458.

  44. Schwerk C, Schulze-Osthoff K . (2005). Mol Cell 19: 1–13.

  45. Seddon B, Legname G, Tomlinson P, Zamoyska R . (2000). Science 290: 127–131.

  46. Sepp-Lorenzino L, Ma Z, Lebwohl DE, Vinitsky A, Rosen N . (1995). J Biol Chem 270: 16580–16587.

  47. Singer AL, Koretzky GA . (2002). Science 296: 1639–1640.

  48. Skorski T . (2002). Nat Rev Cancer 2: 351–360.

  49. Sohn D, Schulze-Osthoff K, Janicke RU . (2005). J Biol Chem 280: 5267–5273.

  50. Stepczynska A, Lauber K, Engels IH, Janssen O, Kabelitz D, Wesselborg S et al. (2001). Oncogene 20: 1193–1202.

  51. Straus DB, Weiss A . (1992). Cell 70: 585–593.

  52. Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S et al. (1995). Nature 376: 596–599.

  53. Tang D, Lahti JM, Kidd VJ . (2000). J Biol Chem 275: 9303–9307.

  54. Uckun FM, Waddick KG, Mahajan S, Jun X, Takata M, Bolen J et al. (1996). Science 273: 1096–1100.

  55. von Haefen C, Wieder T, Essmann F, Schulze-Osthoff K, Dörken B, Daniel PT . (2003). Oncogene 22: 2236–2247.

  56. Wang GQ, Gastman BR, Wieckowski E, Goldstein LA, Gambotto A, Kim A et al. (2001). J Biol Chem 276: 34307–34317.

  57. Webb BL, Jimenez E, Martin GS . (2000). Mol Cell Biol 20: 9271–9280.

  58. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M et al. (2000). Genes Dev 14: 2060–2071.

  59. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ et al. (2001). Science 292: 727–730.

  60. Williams BL, Schreiber KL, Zhang W, Wange RL, Samelson LE, Leibson PJ et al. (1998). Mol Cell Biol 18: 1388–1399.

  61. Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI et al. (2005). Genes Dev 19: 1294–1305.

  62. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ . (1997). J Cell Biol 139: 1281–1292.

  63. Zamoyska R, Basson A, Filby A, Legname G, Lovatt M, Seddon B . (2003). Immunol Rev 191: 107–118.

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Acknowledgements

We thank Drs RT Abraham, A Kosugi, I Schmitz and A Weiss for valuable cells and reagents. This work was supported by the Deutsche Krebshilfe.

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Correspondence to K Schulze-Osthoff.

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Samraj, A., Stroh, C., Fischer, U. et al. The tyrosine kinase Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expression. Oncogene 25, 186–197 (2006) doi:10.1038/sj.onc.1209034

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Keywords

  • apoptosis
  • Bak
  • Bax
  • Lck
  • mitochondria

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