The Tyrosine Kinase Lck Is Involved in Regulation of Mitochondrial Apoptosis Pathways

Oncogene (2003) 22, 176–185 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc The tyrosine kinase Lck is involved in regulation of mitochondrial apoptosis pathways Claus Belka*,1,3, Charlotte Gruber1,3, Verena Jendrossek1, Sebastian Wesselborg2 and Wilfried Budach1 1Department of Radiation Oncology, University of Tu¨bingen, Hoppe Seyler Str. 3, D-72076 Tu¨bingen, Germany; 2Department of Internal Medicine I, University of Tu¨bingen, Otfried Mu¨ller Str. 10, D-72076 Tu¨bingen, Germany The induction of apoptosis requires the activation of a of the caspase cascade at the receptor level, DNA highly coordinated signaling network ultimately leading to damage and stress-mediated caspase activation and the activation of caspases. In previous experiments we and apoptosis are triggered mainly by mitochondrial alter- others have shown that the tyrosine kinase Lck is required ations (Belka et al., 2000b, 2001; Newton and Strasser, for adequate apoptosis induction in response to ionizing 2000). radiation, ceramide incubation and overexpression of the Mitochondrial apoptosis is basically regulated by a HIV-TAT protein. However, the position of Lck within complex interplay of pro- and antiapoptotic Bcl-2-like given apoptotic signaling cascades remains unclear. We proteins. DNA damage or cellular stress triggers the therefore aimed to define the role of Lck during radiation- transcriptional activation of the proapoptotic Bax-like induced apoptosis. Apoptosis induction in response to molecules Bax, Bak and the BH3-only proteins Noxa and ionizing radiation, CD95 or TRAIL receptor stimulation Puma. Although the interplay of Bax-like molecules and was determined in Jurkat T-cells, the Lck-deficient BH3-only proteins is not completely understood, it has Jurkat clone JCaM1.6- and Lck-retransfected been shown that an interaction of molecules belonging to JCaM1.6/Lck. No apoptosis, release of cytochrome c, either group is crucial for the subsequent release of breakdown of the mitochondrial potential were detectable cytochrome c (Cheng et al., 2001). The release of during the first 48 h after irradiation of JCaM1.6 cells. In cytochrome c together with dATP and APAF-1 in turn parallel, no activation of caspase-9, -8 and -3 was triggers the autoproteolytic activation of caspases (re- detectable. Since mitochondrial apoptosis pathways act viewed in: Belka and Budach, 2002; Rudner et al., 2002). within a feedback mechanism during death-receptor- In this regard, the role of certain caspases was also mediated apoptosis, the influence of the Lck defect on found to be different. While caspase-8, which is directly CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was activated at the receptor complex, was shown to be the also tested. Both stimuli induced apoptosis in Lck- key caspase for death-receptor-mediated apoptosis deficient cells. However, the kinetics of apoptosis induc- (Varfolomeev et al., 1998), caspase-9 is crucial for tion determined by caspase-8, -9 and -3 activation as well DNA-damage-related apoptosis and is activated follow- as DWm breakdown was slowed. We conclude that the Lck ing cytochrome c release from the mitochondrial deficiency influences early steps during radiation-induced intermembrane space (Hakem et al., 1998; Kuida et al., mitochondrial alterations. 1998; Soengas et al., 1999). In this context, we have shown that the apoptotic process triggered by ionizing Oncogene (2003) 22, 176–185. doi:10.1038/sj.onc.1206103 radiation is delayed, but not abrogated in cells lacking caspase-8 (Belka et al., 2000b). Thus, two distinct pathways are responsible for the induction of apoptosis Introduction in response to death receptor ligation or DNA damage or stress (Belka et al., 2000b; Engels et al., 2000; The apoptotic process is regulated by a wide array of Dunkern et al., 2001; Stepczynska et al., 2001; Tomicic diverse molecules. Depending on the stimulus applied et al., 2002; Wieder et al., 2001). two apoptotic cascades can be distinguished: whereas In addition to the essential role of caspases, it was death receptor stimulation acts via a direct activation shown that the tyrosine kinase Lck is involved in the regulation of apoptosis induced by ionizing radiation (Belka et al., 1999), the HIV-TAT protein (Manna and *Correspondence: C Belka, Department of Radiation Oncology, Aggarwal, 2000)and ceramide (Manna et al., 2000). In University of Tu¨ bingen, Hoppe Seyler Str. 3, Tu¨ bingen D-72076, this regard, it is important that the release of ceramides Germany; from cellular membranes is considered to be a key step E-mail: [email protected], http:www.radiation-research.de during radiation-induced apoptosis in some (Santana 3These two authors share first authorship Received 8 July 2002; revised 30 September 2002; accepted 4 et al., 1996; Pena et al., 2000)but not all cell models October 2002 (Burek et al., 2001). Lck and mitochondrial apaptosis pathways C Belka et al 177 Lck belongs to the src-like tyrosine kinase family and apoptotic signaling. To this end, we analyzed the key is normally required for the adequate propagation of T- steps in apoptosis signaling cascade in Lck-deficient cell receptor signals. After stimulation of the T-cell JCaM1.6 and Lck expressing JCaM1.6 cells upon death receptor complex by the respective antigen in combina- receptor ligation and irradiation. tion with an MHC molecule, Lck becomes activated and is involved in the phosphorylation of critical residues of the TCR complex. These phophorylations are essential Results for downstream signaling events including calcium influx, activation of the transcription factor NFAT Apoptosis induction, Lck function and expression and the mitogenic kinases Erk1 and Erk2 (Straus and Weiss, 1992; Kabouridis et al., 1997; Samelson, 2002). In a first set of experiments apoptosis induction after Using normal Jurkat Tcells, the Lck-deficient Jurkat irradiation of JCaM1.6 and JCaM1.6/Lck cells with subclone JCaM1.6 and Lck-transfected JCaM1.6/Lck 10 Gy was followed over 72 h. As shown in Figure 1a, cells, we could show previously that Lck is also the onset of apoptosis was abrogated in cells lacking Lck important for the regulation of apoptosis in response with no apoptosis being detectable prior to 72 h to ionizing radiation (Belka et al., 1999). JCaM1.6 and (23.9711% in Lck-negative cells vs 71.7712.4%). At JCaM1.6/Lck were initially employed to prove that Lck 96 h apoptotic changes also increased in Lck-deficient is involved in T-cell receptor signal transduction (Straus cells (41.2722.5%). However, a strong difference and Weiss, 1992). This finding is paralleled by similar between the Lck-deficient and the Lck-re-expressing results from B-cells, where the B-cell receptor associated cells (75.3711.6%)remained detectable. src-like kinase BTK is essential for radiation-induced In parallel experiments, the expression levels of Lck apoptosis (Uckun et al., 1996). In this context, the and the function of Lck-mediated signaling pathways kinase domain of BTK was crucial for the apoptotic were tested in Jurkat, JCaM1.6 and JCaM1.6/Lck. response to ionizing radiation. In addition to the Using an antibody against the C-terminus of Lck, the findings on BTK, it was shown that activation of Lck expression of Lck was absent in JCaM1.6 cells, while by CD19 crosslinking promoted radiation-induced comparable levels of Lck were detectable in Jurkat and apoptosis in a radiation-resistant B-lymphoma line JCaM1.6/Lck cells (Figure 1b). The functional integrity (Waddick et al., 1993). Similarly, the src-like kinase of Lck-mediated signaling pathways in the JCaM1.6/ Lyn was suggested to be involved in apoptosis regula- Lck cells was verified by analyzing the capability of the tion in response to UV light in DT40 B-cells (Qin et al., respective cell line to respond to CD3/T-cell receptor 1997). (TCR)stimulation with an activation of Erk1/2 kinases. Since Lck has been shown to be involved in apoptosis The CD3/TCR complex was stimulated using phytohe- induction in response to ceramide, radiation and HIV- magglutinin-L (PHA-L, 50 mg/ml)and activation of TAT protein, we aimed to further define its role for Erk1/2 was detected using activation specific antibodies Figure 1 Radiation-induced apoptosis in Lck-deficient cells. JCaM1.6 are negative for Lck expression and function and do not undergo apoptosis in response to ionizing radiation, whereas Jurkat control cells and retransfected JCaM1.6/Lck cells express functional Lck and are sensitive to irradiation. (a)Jurkat cells, Lck negative JCaM1.6 and retransfected JCaM1.6/Lck cells were exposed to 10 Gy ionizing radiation and apoptosis induction was determined every 12 up to 72 h by flow cytometry. (b)Expression of Lck was verfied by Western blotting using an antibody against the C-terminus of Lck. (c)Downstream signaling of Lck was determined by Erk1/2 activation in response to phytohaemagglutinin stimulation. Data show means7s.d. (n ¼ 3) (a) or one representative of three independent experiments (b + c). Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 178 directed against the active kinase (Figure 1c). After determined. As shown in Figure 2, cleavage of PARP by stimulation of normal Jurkat cells with PHA-L, Erk1/2 caspase-3 was not detectable in JCaM1.6 cells, whereas kinase was rapidly activated as indicated by the it clearly occurred in the Lck-expressing JCaM1.6 cells. detection of phosphorylated Erk1/2. No such activation Although caspase-8 is an activator caspase for was detectable in JCaM1.6 cells, whereas treatment of receptor-mediated apoptosis, recent work indicated that JCaM1.6/Lck cell with PHA-L clearly induced Erk1/2 it acts as executor caspase during radiation or drug- phosphorylation. Taken together, the data prove that induced apoptosis (Belka et al., 2000b; Engels et al., intact Lck is absent in JCaM1.6 cells that do not 2000; Wieder et al., 2001).

Load more