Oncogene (2006) 25, 186–197 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE The Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expression

AK Samraj1, C Stroh1, U Fischer and K Schulze-Osthoff

Institute of Molecular Medicine, University of Du¨sseldorf, Germany

Tyrosine of the Src family have been implicated in (Skorski, 2002). In T-, tyrosine key biological processes. Here, we provide evidence that kinases play an integral role in the activation of cells p56Lck, a lymphoid-specific Src kinase, is involved in the through various immunoreceptor molecules, such as the activation of the mitochondrial apoptosis pathway. Lck- T- receptor (TCR)/CD3 complex, CD2, LFA-1 and deficient T cells were completely resistant to anticancer MHC class I (Singer and Koretzky, 2002; Zamoyska drugs. In contrast, apoptosis sensitivity to death receptors et al., 2003). Engagement of the TCR leads to a rapid was not altered, indicating a specific interference of Lck rise in intracellular protein tyrosine , with the mitochondrial pathway. Re-expression of Lck followed by a series of biochemical events that result in restored sensitivity to drug-induced apoptosis and trig- effector function in T cells. An initial TCR signal is gered mitochondrial cytochrome c release and caspase mediated by the activation of the Src family tyrosine activation. Further analysis identified that the sensitiza- kinases Lck and Fyn, which phosphorylate specialized tion by Lck was independent of classical mediators of motifs in the signal-transducing subunits of the TCR T-cell signaling, but essentially involved the Bcl-2 protein complex. Phosphorylation of these immunoreceptor Bak. Expression of Bak was completely absent in Lck- tyrosine-based activation motifs creates docking sites deficient cells, while re-expression of Lck transcriptionally for another , ZAP-70. As a result of triggered Bak expression and conferred sensitivity to ZAP-70 recruitment, the TCR is effectively endowed apoptosis, associated with a proapoptotic conformational with kinase function, leading to the activation of a series change of Bak. Furthermore, in vitro the truncated of second-messenger cascades (Kane et al., 2000). These fragment of Bid specifically activated Bak and cyto- include, among others, , the phospha- chrome c release only from mitochondria of Lck- tidylinositol pathway, GTPases, MAP kinase cascades expressing cells. These results do not only demonstrate a and proinflammatory transcription factors. sentinel role of Lck in drug resistance but also delineate a A few recent studies indicate that tyrosine kinases hitherto unknown pathway of Src kinases in regulation of might not only play a role in T-cell activation but also in Bcl-2 . apoptosis. During activation-induced cell death, Lck has Oncogene (2006) 25, 186–197. doi:10.1038/sj.onc.1209034; been implicated in the induction of CD95 ligand published online 22 August 2005 expression (Gonzalez-Garcia et al., 1997). Furthermore, studies using Lck-deficient T-cell lines have suggested Keywords: apoptosis; Bak; Bax; Lck; mitochondria 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 Introduction Lyn have been implicated in apoptosis in response to radiation and growth factor deprivation (Uckun et al., Nonreceptor protein tyrosine kinases exert crucial roles 1996; Qin et al., 1997; Hernandez-Hansen et al., 2004). in a variety of cellular processes including cell growth, The mechanism of how activation of Lck or related differentiation, apoptosis and gene transcription. Tyr- protein kinases contributes to apoptosis in certain osine kinases normally function as tightly regulated situations, while inducing proliferation in others, is switches in the signal transduction network of the cell. If unknown. these switches become stuck in the ‘on’ position, they Apoptotic signaling cascades can be divided into two have the potential to induce oncogenic transformation major pathways, both of which depend on the formation of large multiprotein complexes and the activation of Correspondence: Dr K Schulze-Osthoff, Institute of Molecular caspases (Schulze-Osthoff et al., 1998; Schwerk and Medicine, University of Du¨ sseldorf, Building 23.12, Universita¨ tsstrasse Schulze-Osthoff, 2005). Upon formation of the death- 1, Du¨ sseldorf 40225, Germany. inducing signaling complex in the extrinsic death E-mail: [email protected] 1Both these authors contributed equally to this work. receptor pathway or the apoptosome in the intrinsic Received 15 June 2005; revised 18 July 2005; accepted 25 July 2005; mitochondrial pathway, caspase-8 or -9 is proteolyti- published online 22 August 2005 cally processed resulting in the activation of downstream Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 187 caspases and cleavage of numerous substrates (Fischer mitochondrial membrane, Bax typically resides in the et al., 2003; Schwerk and Schulze-Osthoff, 2003). cytosol or is only loosely associated with membrane Intrinsic pathway stimuli, including many agents that surfaces (Wolter et al., 1997). elicit cellular stress, require the release of mitochondrial In the present study, we investigated the role of the proteins, such as cytochrome c and Smac/Diablo, for Lck kinase in apoptosis. We found that Lck-deficient caspase activation, whereas extrinsic pathway signals Jurkat T cells were completely resistant to mitochon- activate caspases more directly. Bcl-2 proteins function drial apoptosis triggered by anticancer drugs, whereas at a critical checkpoint in the intrinsic pathway to the sensitivity to the death receptor pathway was largely regulate mitochondrial membrane permeability and unaffected. Furthermore, we identified that the block of release of cytochrome c (Ferri and Kroemer, 2001). apoptosis by Lck deficiency was caused by the selective Two major groups of Bcl-2 family proteins exist: the failure to express the proapoptotic protein Bak, even prosurvival members such as Bcl-2, Bcl-xL, Bfl-1/A1, though the cells remained proficient for Bax. Re- Bcl-w, Mcl-1 and Boo, and the proapoptotic members expression of Lck reconstituted Bak expression, caspase including Bax, Bak and Bok (Cory et al., 2003; Daniel activation and, moreover, restored the capability of et al., 2003). Three-dimensional imaging suggests that purified mitochondria to release cytochrome c in both pro- and antiapoptotic Bcl-2 family members share response to Bid treatment. Thus, our results identify a common structure including three or four regions, signal transduction by Lck kinase as a novel pathway called the Bcl-2 homology (BH)domains. Bcl-2 proteins that controls Bak expression. Despite overlapping interact with another group of proapoptotic proteins functions of Bak and Bax, our results also show that that share at least one common motif with Bcl-2, namely Lck-controlled Bak expression is required for the the BH3 domain. BH3-only members include, for mitochondrial apoptotic pathway and thus may con- instance, Bad, Bim, Bid, Bik/Nbk, Puma and Noxa tribute to chemoresistance of human leukemic cells. (Puthalakath and Strasser, 2002). Some BH3-only proteins selectively interact with antiapoptotic Bcl-2 family members, whereas others also interact with Results proapoptotic members. BH3-only proteins have been proposed to act as allosteric regulators of the Bcl-2 Deficiency of the Lck tyrosine kinase results in resistance proteins and as sensors of apoptotic signaling. For to apoptosis induced by anticancer drugs example, during death receptor-mediated apoptosis Bid T-cells express primarily two members of the Src family is cleaved into the death-promoting p15 fragment, tBid, of tyrosine kinases, Lck and Fyn, both of which have which is targeted to mitochondria and binds to either been implicated in various biological processes. To Bax or Bak, resulting in their activation. examine the role of Lck in apoptosis, we used the The generation of Bax and Bak knockout mice has JCaM1.6 cell line, a genetic variant of Jurkat T cells elucidated how proapoptotic Bcl-2 proteins could deficient in p56Lck protein because of a deletion of exon 7 induce cell death. Cells lacking both Bax and Bak do encoding its ATP (Straus and Weiss, 1992). not die in response to various apoptotic stimuli As revealed by immunoblot analysis, an antibody against including BH3-only proteins, in contrast to cells the N-terminus of Lck detected small amounts of a expressing either one of these molecules (Wei et al., 49 kDa splice product in JCaM cells (Figure 1a), whereas 2001). This provided the insight that BH3-only proteins an antibody against the C-terminus did not show any require at least one Bax-type partner to induce cell immunoreactivity (data not shown). Both the parental death. Both Bax and Bak undergo a conformational and Lck-transfected JCaM cells expressed change in response to apoptotic stimuli, which mediates similar amounts of the wild-type kinase at 56 kDa. exposure of their occluded N-terminus (Desagher et al., Analysis of apoptosis showed that, in contrast to 1999; Griffiths et al., 1999). Moreover, they assemble parental Jurkat cells, JCaM cells were almost completely into homomultimers with channel-forming properties in resistant to apoptosis induced by the topoisomerase II the mitochondrial membrane (Antonsson et al., 1997; inhibitor etoposide, even at high drug concentrations Wei et al., 2000). The conformational change of Bak or and after prolonged incubation for 48 h (Figure 1b). Bax is inducible by BH3-only proteins and inhibitable Apoptosis resistance of JCaM cells was not only by Bcl-2. The model emerging from these findings is that observed after treatment of cells with a topoisomerase cytochrome c release depends on interaction between a inhibitor but also after incubation with unrelated anti- BH3-only protein and a Bax-type partner, which allows cancer drugs including doxorubicin, daunorubicin and formation of a Bax-type transmembrane pore. Bak and mitomycin C. Interestingly, re-expression (Figure 1b)of Bax are generally assumed to be able to substitute for Lck in JCaM cells fully overcame drug resistance and each other, since deficiency for both genes is required to reconferred apoptosis sensitivity in response to all render cells completely resistant to apoptosis. However, chemotherapeutic agents. recent evidence also suggests that Bax and Bak exert JCaM cells were also resistant to apoptosis induced nonredundant roles and differentially regulate cell death by staurosporine, a classical inducer of the mitochon- (Panaretakis et al., 2002; Cartron et al., 2003; Gillissen drial apoptosis pathway, and again re-expression of Lck et al., 2003; Klee and Pimentel-Muinos, 2005). Bax and sensitized the cells to apoptosis (Figure 1c and d). The Bak also differ in their initial cellular location. While resistance of JCaM cells was in a similar range as in Bak is constitutively integrated into the ER or outer Jurkat cells overexpressing Bcl-2. However, regardless

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 188

Figure 1 Lck-deficient Jurkat cells are resistant to mitochondrial apoptosis. (a)Expression of p56 Lck 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 mg/ml), daunorubicin (1 mg/ml), doxorubicin (1 mg/ml)or mitomycin C (5 mg/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 mg/ml)or staurosporine (2.5 mM), 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.

of the Lck status, both Lck-deficient and -proficient cells against drug-triggered, but not CD95-mediated apop- were almost equally sensitive to death receptor CD95 tosis therefore indicated that Lck interfered with the ligation when measured after 24 h (Figure 1d); a slight mitochondrial pathway of apoptosis. protection was only seen at early time points (Figure 1c). Similarly, CD95-mediated apoptosis was only delayed Lck inhibits the mitochondrial caspase cascade but not prevented in Bcl-2-overexpressing Jurkat cells. The mitochondrial pathway is triggered by the release The selective protection contributed by Lck deficiency of cytochrome c, which together with Apaf-1 binds to

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 189 procaspase-9 to form the apoptosome and results in the activation of caspase-9 was the mitochondrial release of activation of effector caspase-3, -6 and -7. To determine cytochrome c, which was exclusively seen in the presence in more detail the reason for the inability of Lck- of Lck. deficient cells to undergo apoptosis, we monitored the The mitochondrial pathway is connected with the release of cytochrome c from mitochondria and the death receptor pathway by the Bcl-2 protein Bid that, activation of caspase-3 and -9. In Lck-expressing cells, upon cleavage by caspase-8, translocates to the mito- but not in Lck-deficient cells, etoposide treatment chondria to trigger the release of cytochrome c.In resulted in the marked activation of caspase-3 within JCaM/Lck but not in JCaM cells, the truncated p15 3–4 h, as determined by the proteolytic processing from form of Bid began to appear within 4 h after etoposide its 32 kDa precursor into the 17 kDa active subunit treatment (Figure 2b). In addition, cleavage of caspase-8 (Figure 2a). A similar result was obtained when into the p18 subunit was only observed in Lck- activation of caspase-3 was measured by fluorogenic expressing cells. In comparison to cytochrome c release substrate assays or by the cleavage of its substrate and caspase-9 processing, the cleavage of Bid and PARP-1 (data not shown). The cleavage of caspase-3 caspase-8 was delayed, indicating that it occurred by a was preceded by the activation of caspase-9 that was postmitochondrial event, as also suggested by others only observed in Lck-expressing but not in Lck-deficient (Engels et al., 2000; Tang et al., 2000; von Haefen et al., JCaM cells (Figure 2a). Almost coincident with the 2003; Sohn et al., 2005).

Figure 2 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 mg/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.

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 190 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 proapop- totic molecules such as Bad and Forkhead 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). Inhibi- tion 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. Treatment of both cell lines with etoposide also did not affect the activation of Akt (Figure 3a). Lck- Figure 3 Protection against apoptosis in Lck-deficient cells does expressing cells even showed a slightly stronger phos- not involve MAP kinases, Akt or c-Myc. (a)Time course of kinase phorylation of Akt. Moreover, no differences were activation and c-Myc expression in JCaM and JCaM/Lck cells found in the expression levels of c-Myc. Using gel during etoposide-induced apoptosis. Cells were incubated for the retardation analysis, we also investigated the activation indicated times with 5 mg/ml etoposide, before total cell extracts were investigated for the expression of p38, Erk1/2, JNK, Akt and c-Myc of transcription factors AP-1, NF-AT and NF-kB, by immunoblot analysis. Activation of the kinases was measured which might provide a T-cell survival signal. In contrast using phosphospecific antibodies. (b)Effect of kinase inhibitors on to classical proinflammatory stimuli, etoposide treat- etoposide-induced apoptosis in JCaM/Lck cells. Cells were either left ment did not induce a significant activation of the untreated (control)or pretreated with the MEK inhibitor U0126 or the JNK inhibitor SP600125, and then incubated with etoposide. transcription factors nor were there differences between Apoptosis was assessed after 12 h; the results show means of two both cell lines (data not shown). Thus, these results independent experiments measured in triplicate. demonstrate that neither the activation of antiapoptotic transcription factors or Akt nor effects mediated by MAP kinases were involved in the apoptosis resistance resulted in expression levels of Bak similar to wild-type mediated by Lck deficiency. Jurkat cells (Figure 4a). We also compared the expression levels of inhibitor of Lck specifically controls Bak expression apoptosis proteins (IAPs), which directly bind to and The mitochondrial pathway is controlled by the ratio inhibit active caspases. However, expression of neither of pro- and antiapoptotic Bcl-2 proteins. Since Lck c-IAP1 and c-IAP2 nor of XIAP differed in the deficiency resulted in an apoptosis defect probably individual cell lines (Figure 4a), indicating that these upstream of cytochrome c release, we investigated the apoptosis inhibitors were presumably not responsible expression of various proapoptotic Bcl-2 family mem- for the apoptosis-resistant phenotype of Lck-deficient bers, such as Bax, Bak and Bad, and anti-apoptototic cells. proteins, such as Bcl-2 and Bcl-xL. Unexpectedly, We next investigated the subcellular localization of apoptosis-resistant JCaM cells revealed even slightly the Bcl-2 proteins in the course of etoposide treatment reduced levels of Bcl-2 and Bcl-xL compared to parental and therefore prepared cytosolic and mitochondrial Jurkat and Lck-transfected JCaM cells. This might fractions of JCaM cells and their Lck-expressing concur with the observation that Lck stimulates the counterparts. In support of the previous result, cyto- expression of these antiapoptotic molecules. Jurkat chrome c was only released from mitochondria of cells and their Lck-deficient counterparts expressed Lck-expressing but not of Lck-deficient cells. Both Bcl- similar levels of Bax. Striking, however, was our 2 and Bcl-xL were mainly detected in the mitochondrial observation that expression of the Bax-related proa- fraction and remained relatively constant during poptotic Bcl-2 member Bak was almost completely etoposide treatment (Figure 4b). Also, Bax was mainly absent in JCaM cells, whereas re-expression of Lck found at the mitochondria. Importantly, fractionation

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 191

Figure 4 Expression status of Bcl-2, IAP family proteins and NF-kB 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.

in cytosolic and mitochondrial extracts confirmed that Bak, which undergo a N-terminal conformational Bak was strikingly absent in JCaM cells, whereas change resulting in cytochrome c release. The con- considerable levels were detected in mitochondria from formational change of Bak and Bax can be analysed JCaM/Lck cells. In line with the previous data, during using specific antibodies against their normally occluded drug treatment Bak was not translocated from the N-terminus. FACS analysis of JCaM cells with such cytosol but constitutively found in the mitochondrial antibodies revealed only a slight activation of Bax after compartment. etoposide treatment, whereas due to its absence, To investigate whether the differential expression of no conformational change of Bak was observed Bak was also detectable at the mRNA level, we (Figure 6a). In contrast, JCaM/Lck cells showed a performed reverse transcriptase–polymerase chain reac- pronounced conformation change of Bak upon apopto- tion (RT–PCR)analyses. As shown in Figure 5a, in sis induction (Figure 6b). Interestingly, however, under JCaM cells Bak-specific transcripts were almost comple- these conditions JCaM/Lck cells also displayed a tely absent, whereas Bak mRNA was expressed in the stronger activation of Bax compared to the Bak- parental Jurkat and Lck-transfected cells. This expression deficient JCaM cells. This result therefore suggests a pattern did not change when JCaM and JCaM/Lck cells degree of functional cooperation of Bak and Bax were stimulated with etoposide or phytohemagglutinin regarding their conformational activation. (Figure 5b). Stimulation of the cells also did not affect the To further substantiate these findings, we treated expression levels of other pro- and antiapoptotic Bcl-2 mitochondria in digitonin-permeabilized JCaM/Lck proteins. Together, these data show that Lck strongly cells with recombinant tBid, representing the proapop- regulates Bak expression at the transcriptional level. totic, caspase-8 generated form of Bid. The addition of exogenous tBid rapidly induced the conformational Bak undergoes a proapoptotic conformational change in change of Bak in vitro (Figure 7a). The effect was Lck-expressing cells and mediates cytochrome c release associated with the release of cytochrome c into the both in vitro and in vivo supernatant and a corresponding reduction of cyto- Recent data show that mitochondrial apoptosis is chrome c in mitochondria (Figure 7b). The translocation regulated by the multidomain Bcl-2 proteins Bax or of cytochrome c by tBid was specifically observed in

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 192 a JCaM JCaM 200 200

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Figure 5 Transcriptional expression of Bcl-2 family members in 0 0 100 101 102 103 104 100 101 102 103 104 Lck-deficient and -proficient Jurkat cells. (a)RNA from JCaM, FL1-H FL1-H JCaM/Lck and parental Jurkat cells was subjected to RT–PCR using oligonucleotide primers specific for Bax, Bak, Bcl-2 and Bcl- Figure 6 Requirement of Lck for Bak and Bax conformational x. The PCR products were separated on agarose gels and visualized change. JCaM (a)and JCaM/Lck cells ( b)were either left untreated by ethidium bromide staining. The closed arrowheads indicate the or incubated for 4 h with etoposide. The conformational change relevant PCR products and the open arrowheads a GAPDH PCR was measured by flow cytometry using activation-specific anti- product that was amplified in parallel. (b)Expression of Bcl-2 bodies against Bax and Bak. The filled histograms show the family members in JCaM/Lck and JCaM cells during phytohe- staining with isotype-matched control antibodies and the grey line magglutinin (PHA)and etoposide treatment. Cells were treated for indicates the specific staining for the active forms of Bak and Bax. the indicated time with PHA (50 mg/ml)or etoposide (5 mg/ml), before RNA was extracted and subjected to RT-PCR analysis. stimulation by anti-CD3, the activation of Lck by etoposide was slightly delayed but clearly visible. JCaM/Lck cells, but virtually absent in mitochondria Furthermore, treatment of cells with the Src kinase from Lck-deficient cells. These in vitro data therefore inhibitor herbimycin A, which targets Lck for protea- confirm that the defect of the apoptosis-resistant and somal degradation (Sepp-Lorenzino et al., 1995), Lck-deficient cells is directly located at the mitochondria strongly prevented apoptosis induction by etoposide and mediated by Bak deficiency. (Figure 8b). Surprisingly, however, the pyrazolopyrimi- dine compound PP2, which is a direct inhibitor of the kinase activity of Lck (Hanke et al., 1996), was T-cell signaling and kinase activity of Lck are not completely ineffective in inhibiting cell death, even when required for apoptosis high concentrations were used (Figure 8b). This result It was reported that some biological effects of Lck therefore indicates that the kinase activity is not require different structural domains (Denny et al., 1999). required for the proapoptotic effect of Lck. We therefore investigated the specificity of the pro- During T-cell stimulation, Lck phosphorylates several apoptotic effect of Lck and its downstream signaling. signaling mediators, most importantly ZAP-70, which Treatment of JCaM/Lck cells with etoposide resulted then phosphorylates key adapter proteins, including in the activation of Lck as measured by its auto- LAT (linker for activation of T cells). Loss of ZAP-70 phosphorylation (Figure 8a). In comparison to TCR or LAT disrupts TCR signaling and blocks T-cell

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 193 activation (Finco et al., 1998; Williams et al., 1998). To or JCaM/Lck cells, and neither was apoptosis sensitivity analyse whether these key mediators of T-cell signaling impaired in ZAP-70-deficient Jurkat cells. Moreover, were involved in the proapoptotic effect of Lck, we used apoptosis was also not blocked in JCaM cells retrans- ZAP-70- and LAT-deficient Jurkat cells (Figure 8c). fected with a -deficient Lck construct, However, cells deficient for LAT underwent apoptosis indicating that Lck myristoylation, which is essential for to a similar extent as their LAT-transfected counterparts T-cell stimulation, was not required for apoptosis sensitization. In addition, neither cells expressing the myristoylation-deficient Lck nor cells deficient for a 200 200 Control tBID ZAP-70 or LAT showed defects in Bak expres- 160 160 sion (Figure 8d), suggesting that different signals 120 120 are required for T-cell stimulation and the apoptosis-

Counts 80 80 Counts sensitizing effect of Lck.

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b JCam JCam/Lck Co tBid Co tBid

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Figure 7 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 mM 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 mM 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.

Figure 8 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 mM herbimycin A or 20 mM 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 value7s.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 mean7s.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.

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 194 Discussion To investigate the mechanism of chemoresistance in Lck-deficient cells, we investigated several pathways The present study demonstrates a critical role of Lck for involved in apoptosis regulation. Inhibitors of stress- induction of apoptosis in human leukemic cells in responsive kinases including Erk, p38 and JNK did not response to anticancer drugs. The deficiency in Lck affect apoptosis sensitivity of Jurkat cells in response endowed Jurkat cells with a potent resistance mechan- to various chemotherapeutic drugs. Furthermore, no ism to various stimuli that activate the mitochondrial differences in the activation profile of Akt or the pathway, whereas it did not block apoptosis triggered by transcription factors NF-AT, AP-1 and NF-kB were death receptors. Moreover, apoptosis resistance was observed between Lck-deficient and -proficient cells, and completely reversed in cells with reconstituted Lck neither were the expression levels of various IAP expression. We also demonstrate that the apoptosis- members altered, suggesting that none of these classical resistant phenotype of Lck-deficient cells is caused by apoptosis pathways was involved in the resistance the selective defect of Bak expression, suggesting that caused by the lack of Lck. Lck is not only a mediator of chemosensitivity but also a We found that the block in the apoptotic cascade in hitherto unknown regulator of Bak expression. Lck-deficient cells was rather localized to the mitochon- At first view, it appears surprising that Lck which has drial apoptosis machinery. No cytochrome c release or been mainly linked to T-cell activation regulates caspase activation was detected in Lck-deficient cells apoptosis. However, the proliferation of cells requires treated with chemotherapeutic drugs. Furthermore, in not only mitogenic signals but also survival signals, contrast to mitochondria from wild-type cells or cells which provide a safeguard against deregulated prolif- reconstituted with Lck, those from Lck-deficient cells eration. Thus, mitogenic signaling without accompany- did not release cytochrome c in response to tBid in vitro. ing survival signals triggers apoptosis and leads to the This observation indicates that the apoptotic defect of elimination of potentially malignant cells. The onco- Lck-deficient cells was directly localized at mitochon- genes c-Myc and E2F, for example, which generate dria. Investigating the expression of Bcl-2 family strong proliferative signals, induce apoptosis in the members, we found basically no differences in the absence of exogenous survival factors (Green and Evan, expression levels of several anti- or proapototic proteins. 2002). Nonreceptor tyrosine kinases of the Src family However, Bak, which in addition to Bax is considered might also exert such a dual role in proliferation and as the gatekeeper of cytochrome c release, was conspi- apoptosis. Lck appears to be required for ceramide-, cuously absent in Lck-deficient cells. Importantly, drug- and irradiation-induced apoptosis (Belka et al., reconstitution of Lck fully restored Bak expression 1999; Manna et al., 2000; Gruber et al., 2004), while the to a similar level as in wild-type cells. B-cell receptor-associated Src-like kinases Btk and Lyn The critical role of Bak in Lck-mediated apoptosis have been implicated in apoptosis in response to sensitization was substantiated by experiments with radiation (Uckun et al., 1996; Qin et al., 1997; conformation-specific antibodies that allow the mea- Hernandez-Hansen et al., 2004). Interestingly, periph- surement of Bak and Bax activation. Interestingly, after eral T cells from mice with inducible Lck deficiency were treatment of JCaM cells with genotoxic agents, a weak reported to have prolonged survival in vivo, although the conformational change of Bax occurred, whereas JCaM/ role of altered apoptosis has not been addressed in this Lck cells displayed a stronger activation of Bax study (Seddon et al., 2000). Also c-Src and v-Src can compared to the Bak-deficient JCaM cells. As pre- sensitize cells to apoptosis (Webb et al., 2000; Melendez viously reported (Mikhailov et al., 2003; Lindenboim et al., 2004). Recently, it has been demonstrated that et al., 2005), this might indicate a functional cooperation overexpression of two Drosophila Src kinases not only between Bak and Bax with regard to their conforma- induces ectopic cell proliferation but also apoptosis in tional activation. It has been proposed that tBid acts transgenic flies (Pedraza et al., 2004). Src-induced partly by inducing conformational changes in Bax proliferation and apoptosis were found to be largely (Eskes et al., 2000). However, tBid can also trigger independent events, as blocking ectopic proliferation did cytochrome c release also independently of Bax (Desa- not block cell death in Drosophila. gher et al., 1999). Furthermore, Bak-deficient hepato- Our study demonstrates that the apoptotic role of Lck cytes and T cells are resistant to tBid induction of is also independent of its principal function in T-cell cytochrome c release (Wei et al., 2000; Wang et al., signaling as well as its kinase activity. Following TCR 2001). This therefore suggests that at least in certain cell engagement, Lck mediates phosphorylation ZAP-70 and types, Bak rather than Bax is the key regulator of LAT, and ultimately promotes the activation of down- mitochondrial apoptosis and might determine the stream pathways. Using Jurkat clones deficient in these chemoresistance of tumor cells. T-cell mediators, we clearly show that ZAP-70 and LAT Bak and Bax are generally assumed to substitute for are not involved in Lck-mediated apoptosis resistance. each other, since deficiency for both genes is required to Moreover, myristoylation, which is essential for target- render cells resistant to a number of proapoptotic ing Lck to the plasma membrane during T-cell stimula- agents, while single knockouts for either gene have far tion, was not required, as JCaM cells reconstituted with less effects. Bak and Bax single knockout mice are rather a myristoylation-deficient Lck mutant were equally normal, whereas most of the mice lacking both genes sensitive to apoptosis as parental Jurkat cells or cells display multiple developmental defects (Lindsten et al., reconstituted with wild-type Lck. 2000), suggesting that Bax and Bak have overlapping

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 195 roles in the regulation of apoptosis. These overlapping deficient Jurkat cell clone JCaM1.6 (Straus and Weiss, 1992) roles, which were observed in murine models for and Lck-retransfected JCaM1.6 cells as well as JCaM2 cells development of normal cells, remain to be elucidated deficient for LAT (Finco et al., 1998)and its retransfected in human tumor cells. Despite their regulation by p53, derivatives were kindly provided by A Weiss (San Francisco, Bak and Bax also have proapoptotic effects in cells with USA). Jurkat cells deficient for ZAP-70 (Williams et al., 1998) were a gift from RT Abraham (Durham, NC, USA). JCaM1.6 altered p53 expression (Knudson et al., 2001; Degen- cells stably transfected with a myristoylation-deficient Lck hardt et al., 2002). Therefore, a redundant function for construct were obtained from A Kosugi (Osaka, Japan). Bax and Bak, as detected in normal murine cells, may be Jurkat cells overexpressing Bcl-2 have been described pre- differentially regulated in cancer cells with mutated p53, viously (Stepczynska et al., 2001). Staurosporine, etoposide, such as human leukemic cells. It is possible that in tumor doxorubicin, daunorubicin, mitomycin C and propidium cells the balance between Bax and Bak levels of iodide were purchased from Sigma (Deisenhofen, Germany). expression is different from that of normal cells. Agonistic anti-CD95 and caspase-8 moAb were from Bio- Moreover, although Bak and Bax might have over- Check (Mu¨ nster, Germany). Anti-Bid, anti-caspase-3 moAb lapping functions, it has been shown that they may serve and polyclonal anti-caspase-9 were from R&D Systems different roles depending on the proapoptotic stimulus (Wiesbaden, Germany)and New England BioLabs (Beverly, MA, USA), respectively. Anti-Bax and cytochrome c moAbs (Panaretakis et al., 2002; Cartron et al., 2003). For were from PharMingen (Hamburg, Germany). The conforma- instance, induction of cell death by the BH3-only tion-specific antibodies rabbit anti-Bax-NT and monoclonal protein Nbk/Bik is entirely Bax dependent, but does anti-Bak were from Upstate Biotechnology (Lake Placid, NY, not require Bak (Gillissen et al., 2003). It is also USA)and Oncogene, respectively. MoAbs against Lck, actin, conceivable that Bak and Bax could be sequestered by Bcl-2 and c-Myc, and antisera against c-IAP1 and c-IAP2 w different antiapoptotic Bcl-2 members that are targeted ere purchased from Santa Cruz Biotechnology (Santa Cruz, by distinct BH3 proteins (Willis et al., 2005). CA, USA). Anti-Tom20 MoAb and polyclonal anti-Bcl-xL Lck deficiency resulted in a lack of Bak expression at were from Transduction Laboratory (Heidelberg, Germany). the mRNA level, suggesting Lck regulates transcrip- Anti-JNK, anti-p38 MAPK, anti-p44/42 (Erk-1), and anti-Akt tional activation of the Bak gene. So far, transcriptional were obtained from Cell Signaling. Activation of the kinases was monitored with anti-phospho-p38 (T180/Y182)and regulation of most Bcl-2 proteins is poorly understood. anti-phospho-Akt (Y326)rabbit antisera as well as with Various proapoptotic Bcl-2 members including Bax, mouse anti-phospho-SAPK/JNK (T183/Y185)antibody. Puma, Noxa and Bid are p53 target genes in the DNA Anti-phospho-p44/42 Erk (T202/Y204)moAbs were obtained damage response pathway. However, Jurkat cells lack from Cell Signaling. functional p53 (Cheng and Haas, 1990; Iwamoto et al., 1996), indicating that Lck-mediated regulation of Bak Treatment of cells and measurement of cell death expression is p53 independent. In contrast to Bax, the Cells were exposed to anti-CD95 (1 mg/ml)or the drugs transcriptional regulation of Bak has not yet been staurosporine (2.5 mM), etoposide (5 mg/ml), doxorubicin (1 mg/ analysed at the promoter level. Computer analysis of its ml), daunorubicin (1 mg/ml)and mitomycin C (5 mg/ml). 3.5 kb upstream region of the Bak gene reveals no Inhibitors obtained from Calbiochem were used at the TATA-box, but putative binding sites for NF-kB, SP1 following concentrations: SP600125 (1 mM), UO126 (10 mM), and p53 (data not shown). Moreover, an ISRE and PP2 (20 mM)and herbimycin A (10 mM). At the concentrations GAS site is present that could bind IRF and STAT used, the inhibitors did not elicit any cytotoxic response on 4 family members. The latter sites are particularly inter- their own. To determine the level of apoptosis, 3 Â 10 cells/ esting, as they might be at least partially responsible for well were seeded in microtiter plates and treated for the indicated time with anti-CD95 or the chemotherapeutic agents. the apoptosis resistance conferred by targeted gene The leakage of fragmented DNA from apoptotic nuclei was deletion of certain IRF and STAT family members measured as described (Essmann et al., 2003). Briefly, (Tamura et al., 1995; Battle and Frank, 2002). apoptotic nuclei were prepared in a hypotonic lysis buffer In summary, our data indicate a hitherto unrecog- (1% sodium citrate, 0.1% Triton X-100, 50 mg/ml propidium nized link between the tyrosine kinase Lck, Bak iodide)and analysed by flow cytometry. Nuclei to the left of expression and chemoresistance. Although mutations the 2N peak containing hypodiploid DNA were considered as in the Bak gene were detected in human gastric and apoptotic. All flow cytometric analyses were performed on a colorectal cancers (Kondo et al., 2000), Bak deficiency FACScalibur (Becton Dickinson). as a mechanism for chemoresistance has not yet been addressed. The resistance to anticancer drugs suggests Flow cytometric analysis of Bak and Bax conformational that the deficiency of Lck and a consequent failure to changes express Bak participates in the regulation of mitochon- At the indicated times after drug treatment, cells (1 Â 106)were drial apoptosis in leukemic cancer cells. 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 Materials and methods and Bak and isotype-matched control antibodies was per- formed with an 1:50 dilution of the respective antibody in 50 ml Cell lines and reagents staining buffer (PBS, 1% FCS, 50 mg/ml digitonin). Then, cells All cell lines were cultured in RPMI-1640 supplemented with were washed three times and resuspended in 50 ml staining 10% fetal calf serum (FCS), 100 U of penicillin/ml and 0.1 mg buffer containing 0.1 mg Alexafluor 488-labeled chicken anti- streptomycin/ml (PAA Laboratories, Linz, Austria). The Lck- mouse or FITC-coupled goat anti-rabbit IgG (Molecular

Oncogene Lck regulates Bak expression and mitochondrial apoptosis AK Samraj et al 196 Probes)and incubated on ice for 30 min in the dark. After resulting supernatant and mitochondrial pellets were analysed three washing steps, conformational changes of Bak and Bax by immunoblotting. were immediately measured in the FL-1 channel of a flow cytometer. Immunoblotting Proteins (30 mg/lane)were separated under reducing conditions Preparation of cytosolic, mitochondrial and total cell extracts on 10–15% SDS–PAGE and electroblotted to a polyvinylidene After drug treatment, cells were washed in PBS and suspended difluoride membrane. Membranes were blocked for 2 h with in low-salt lysis buffer containing 10 mM HEPES, pH 7.9, 5% milk powder in Tris-buffered saline, and then incubated 1 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithio- overnight at 4 C with the primary antibodies as recommended threitol (DTT), 2 mM phenylmethylsulfonyl fluoride (PMSF) by the manufacturers. Membranes were washed four times and 3 mg/ml of each aprotinin, leupeptin and pepstatin A. with TBS/0.02% Triton X-100 and incubated with the After 20 min on ice, cells were lysed by passing them three respective peroxidase-conjugated secondary antibody for 1 h. times through a 26 G needle. Lysates were then centrifuged at Following extensive washing, the reaction was developed by 500 g for 10 min to remove nuclei and intact cells. The enhanced chemiluminescent staining. supernatants were centrifuged at 10 000 g for 30 min at 41C to obtain a cytosolic fraction. The resulting pellet that Reverse transcriptase–polymerase chain reaction contained crude mitochondria was lysed in high-salt lysis The mRNA expression levels of the different Bcl-2 members buffer (1% NP-40, 20 mM HEPES, pH 7.9, 2 mM PMSF, were monitored by RT–PCR. Total RNA was extracted from 350 mM NaCl, 20% glycerol, 1 mM MgCl2, 0.5 mM EDTA, the cells with TRI reagent (Sigma). RT and PCR were 0.1 mM EGTA, 0.5 mM DTT and protease inhibitors. For total performed using the Titanium One-step RT–PCR (BD cell extracts, cells were directly lysed in a high-salt buffer and Biosciences). For standardization, each RT sample was centrifuged at 10 000 g for 30 min. subjected to PCR for glyceraldehyde-3-phosphate dehydrogen- ase (GAPDH). The 30 and 50 primers used for amplification were 50-ATG TCT CAG AGC AACCCG GAG-30 and 50-TTT Mitochondria isolation CCG ACT GAA GAG TGA GCC C-30 for Bcl-x, 50-GAA To obtain mitochondria for in vitro cytochrome c release GAT CTG CTT CGG GGC AAG GCC CAG GT-30 and 50- assays, cells were permeabilized in buffer A containing 50 mg/ml AAG GAT CCT CAT GAT TTG AAG AAT CTT CG-30 for digitonin, 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl, Bak, 50-GAA GAT CTG ACG GGT CCG GGG AGC AGC pH 7.4, 1 mM DTT and protease inhibitors, and immediately CC-30 and 50-AAG GAT CCT CAG CCC ATC TTC TTC passed three times through a 26 G needle. After centrifugation CAG AT-30 for Bax, 50-ATG GCG CACGCT GGG AGA AC- at 500 g for 10 min, the remaining supernatants were further 30 and 50-CCA GCC TCC GTT ATC CTG GATC-30 for Bcl-2, 1 centrifuged at 10 000 g for 30 min at 4 C. The resulting crude and 50-GTG GAA GGA CTC ATG ACC ACA G-30 and 50- mitochondrial pellets were washed three times with buffer B CTG GTG CTC AGT GTA GCC CAG-30 for GAPDH. containing 250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4, to remove microsomal contaminations. The crude Lck kinase assay mitochondrial fraction was then resuspended in 10 mM Cells were treated for the indicated time with etoposide or anti- HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM CD3 (OKT3), and then extracted in a buffer containing 20 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 4.2 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 1% Nonidet sodium succinate, 1 mM DTT and protease inhibitors, and used for analysis of cytochrome c release (Bantel et al., 2001). P-40, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mM PMSF and 1mM DTT. Cell extracts were immunoprecipitated with 1 mg anti-Lck for 12 h at 41C followed by incubation with protein A Purification of recombinant tBid and in vitro cytochrome c sepharose beads for 1 h. The beads were washed with lysis release assay buffer and kinase buffer (50 mM HEPES, pH 7.4, 20 mM NaCl, The caspase-8-generated fragment of Bid was cloned in 10 mM MgCl2, 100 mM Na3VO4, 500 mM DTT, 25 mM b- PGEX-2T, a bacterial expression vector for glutathione S- glycophosphate), and then assayed in kinase buffer containing fusion proteins. After induction of protein expres- 10 mCi g[32P]ATP and 2.5 mM ATP at 371C for the indicated sion, tBid was purified by glutathione sepharose 4B beads times. Reactions were stopped by the addition of sample buffer (Amersham Bioscience, Uppsala, Sweden). The purified and subjected to SDS–PAGE. fractions were dialysed in PBS and examined for homogeneity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis Acknowledgements (SDS–PAGE). Purified mitochondria from JCaM and JCaM/ We thank Drs RT Abraham, A Kosugi, I Schmitz and A Weiss Lck cells were incubated with GST-tBid at 371C for 30 min. for valuable cells and reagents. This work was supported by Then, the samples were centrifuged at 10 000 g for 20 min. The the Deutsche Krebshilfe.

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