MEKK1-induced apoptosis requires TRAIL death receptor ... - Nature

Oncogene (2002) 21, 6649 – 6656 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

MEKK1-induced apoptosis requires TRAIL death receptor activation and is inhibited by AKT/PKB through inhibition of MEKK1 cleavage Andrea H Bild1, Francisco J Mendoza3, Erika M Gibson3, Mei Huang2, Jacylyn Villanueva3, Timothy P Garrington1, Richard Jove2, Gary L Johnson1 and Spencer B Gibson*,3 1

Department of Pharmacology, University of Colorado, 2400 East Ninth Street, Denver, Colorado, CO 80262, USA; 2Molecular Oncology Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida, FL 33612, USA; 3 Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0V9

MEK kinase 1 (MEKK1) induces apoptosis through the activation of caspases. The mechanism for MEKK1induced apoptosis involves caspase-mediated cleavage of MEKK1, releasing a pro-apoptotic 91 kDa kinase fragment that serves to further amplify caspase activation in a feedback loop. Both cleavage of MEKK1 and increased expression of death receptor 4 (DR4, TRAILR1) and death receptor 5 (DR5, TRAILR2) occur following exposure of cells to genotoxins. Overexpression of kinase inactive MEKK1 inhibits MEKK1-mediated apoptosis and effectively blocks death receptor upregulation following etoposide treatment. Herein, we investigate the role of death receptor activation and the ability of AKT/PKB (AKT) to inhibit cell death in MEKK1-induced apoptosis. We show that by preventing DR4 and DR5 activation through expression of decoy receptor 1 (DcR1) and dominant negative FADD, we inhibit MEKK1-induced apoptosis. Furthermore, expression of 91 kDa MEKK1 increased DR4 and FAS mRNA and protein levels. MEKK1-induced apoptosis is amplified by blocking PI-3 kinase activation and overexpression of AKT blocked both MEKK1-induced apoptosis and caspase activation. AKT overexpression also prevented the cleavage of endogenous MEKK1 by genotoxins. AKT did not, however, block MEKK1-induced JNK activation, showing that regulation of the JNK pathway by MEKK1 is independent of its role in regulation of apoptosis. Thus, MEKK1-induced apoptosis requires TRAIL death receptor activation and is blocked by AKT through inhibition of MEKK1 cleavage. Oncogene (2002) 21, 6649 – 6656. doi:10.1038/sj.onc. 1205819 Keywords: apoptosis; serine threonine kinases; signal transduction; death receptors; caspases; MAPK Introduction MEK kinase 1 (MEKK1) is a 196-kDa mitogenactivated protein kinase (MAPK) kinase kinase that

*Correspondence: SB Gibson; E-mail: [email protected] Received 26 November 2001; revised 19 June 2002; accepted 28 June 2002

participates in regulation of the c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) pathways (Lange-Carter et al., 1993; Widmann et al., 1999). MEKK1 is also involved in induction of apoptosis through the activation of caspases (Widmann et al., 1997, 1999). Exposure of cells to stresses, such as genotoxins, activates caspase 3-like proteases, which cleave MEKK1 into a pro-apoptotic 91 kDa kinase domain fragment (Widmann et al., 1998). This cleavage of MEKK1 releases its 91 kDa form from the membrane fractions into the cytoplasm (Deak et al., 1998; Schlesinger et al., 1998). The 91 kDa kinase fragment further amplifies caspase activation in a feedback loop and is a strong inducer of apoptosis (Cardone et al., 1997; Widmann et al., 1998). Inhibitors of caspases block this cleavage, and mutation of the consensus caspase 3-like cleavage site of MEKK1 inhibits both its cleavage and its ability to induce apoptosis. Both cleavage of MEKK1 and increased expression of DR4 and DR5 occur following exposure of cells to genotoxins (Gibson et al., 2000). Overexpression of a kinase inactive MEKK1 inhibits MEKK1-mediated apoptosis and effectively blocks death receptor upregulation following etoposide treatment (Gibson et al., 2000). Thus, cleavage of MEKK1 serves to activate a cell death-promoting response. MEKK1 is emerging as an important mediator of apoptosis in human cancers. MEKK1 is necessary for the induction of apoptosis following anoikis and specific genotoxic treatments (Cardone et al., 1997; Gibson et al., 1999). MEKK1 is also involved in apoptosis of colon cancer cells following chemotherapy (Soh et al., 2001). Furthermore, aberrant MEKK1 cleavage and subsequent apoptosis in certain ovarian adenocarcinomas leads to drug resistance (Gebauer et al., 2000). Dysregulation of apoptosis can also occur by mutation of genes involved in the death receptor pathways. Death receptors such as DR4, DR5, Fas, and tumor necrosis factor (TNF) receptor belong to the superfamily of TNF receptors that initiate apoptotic signals upon ligation. Activation of these death receptors leads to recruitment of other proteins, including FADD (an adaptor protein) and caspase 8. Association of adaptor proteins with the death receptor

AKT blocks MEKK1 induced apoptosis AH Bild et al

6650

leads to caspase 8 activation, which in turn leads to the activation of caspase 3-like molecules and eventual apoptotic cell death (Ashkenazi and Dixit, 1999; Golstein, 1997; Green, 2000). Inactivating mutations in DR4 and DR5 have been characterized in certain metastatic breast cancer and non-Hodgkin’s lymphomas (Lee et al., 2001; Shin et al., 2001). Further, Fas mutations occur in specific malignant lymphomas and solid tumors (Mullauer et al., 2001; Straus et al., 2001). Other TNF receptor family members, such as decoy receptor 1 (DcR1), bind to ligands for death receptors but, lacking the cytoplasmic domains to recruit proapoptotic proteins, fail to induce apoptosis (Sheridan et al., 1997). By competitive inhibition, these decoy receptors act to circumvent death receptor activation, thereby negatively regulating death receptor-induced signaling and apoptosis. Another protein that negatively regulates apoptosis is the AKT/PKB (AKT) proto-oncogene. AKT is found upregulated in many cancers, including breast, ovarian, prostate, and pancreatic malignancies (BlumeJensen and Hunter, 2001). AKT mediates cell survival by suppressing apoptosis induced by a variety of apoptotic stimuli, including loss of cell adhesion, growth factor withdrawal, and exposure to genotoxins. The action of AKT-mediated protection occurs both by inhibition of pro-apoptotic proteins, such as caspase 9, BAD, and the Forkhead transcription factor, and by activation of anti-apoptotic proteins, such as the NF-kB and CREB. AKT can also promote cell survival through inactivation of caspase-mediated apoptotic signaling (Datta et al., 1999; Khwaja, 1999). Activation of AKT has been shown to block caspase activation and apoptosis following treatment with many different apoptotic stimuli, including anoikis and genotoxic agents, and therefore potentially contributes to chemotherapy resistance in some forms of cancer. Recent studies suggest a role of AKT in inhibitory modulation of TNF-receptor mediated apoptosis. AKT is also implicated in Fas-mediated apoptosis as Pten +/7 mice show decreased Fasinduced apoptosis (Di Cristofano et al., 1999). Further, AKT regulates the expression of c-FLIP, a caspase 8 dominant negative that inhibits Fas death signals in certain tumor cells (Panka et al., 2001; Tschopp et al., 1998). To further define the mechanism of MEKK1mediated apoptosis, we investigated the role death receptor pathway activation. Transfection studies involving overexpression of DcR1 and dominant negative FADD (FADD DN) show that death receptor signaling is required for MEKK1-mediated apoptosis. Inhibition of PI-3 kinase potentiates MEKK1-induced apoptosis, and expression of AKT blocks initial cleavage of endogenous MEKK1 to its pro-apoptotic 91 kDa form and subsequent MEKK1 caspase activation and apoptosis. Thus, MEKK1-induced apoptosis requires death receptor activation and is blocked by AKT through inhibition of caspase activation, which inhibits cleavage of MEKK1 to its pro-apoptotic 91 kDa form. Oncogene

Results Inhibition of death receptor activation prevents MEKK1-induced apoptosis Decoy receptors compete with specific death receptors for ligand binding (Ashkenazi and Dixit, 1999). Overexpression of DcR1 blocks ligand binding and activation of DR4 and DR5 (Pan et al., 1997; Sheridan et al., 1997). To determine if DR4 and DR5 activation is involved in MEKK1-induced apoptosis, human embryonic kidney (HEK) 293 cells overexpressing MEKK1 were transfected with vector alone or with DcR1 cDNA. The extent of apoptosis was determined by TdT staining in a Tunel assay, as described in the Materials and methods section. HEK-293 cells containing empty vector showed 21% apoptosis when MEKK1 was expressed, compared with 4% apoptosis in cells expressing DcR1 in the presence of MEKK1 (Figure 1a). HEK-293 cells were also transiently transfected with MEKK1 in the absence or presence of FADD DN, which acts to block caspase 8 activation following ligand binding of death receptors, including DR4 and DR5. Expression of MEKK1 alone caused 43% apoptosis. In the presence of FADD DN, however, MEKK1 induced only 20% apoptosis, a

Figure 1 MEKK1-induced apoptosis in HEK-293 cells overexpressing DcR1 and FADD DN. (a) HEK-293 lines stably expressing DcR1 or vector alone were transiently transfected with MEKK1. Forty-eight hours following transfection, the cells were stained for MEKK1 and TdT. The percentage of TdT positive cells expressing MEKK1 determined the percent apoptosis. (b) HEK-293 cells were transiently transfected with MEKK1 in the presence or absence of FADD DN. Per cent apoptosis was determined by acridine orange staining. Control cells were untransfected or transfected with FADD DN alone. Results are representative of three independent experiments


6651

percentage similar to that seen in untransfected cells (Figure 1b). Cells expressing FADD DN without MEKK1 also showed no increase in apoptosis above control levels. These results suggest that MEKK1induced apoptosis involves activation of death receptor signaling pathways. MEKK1 upregulates DR4 and Fas mRNA and protein levels To determine if MEKK1 directly affects the mRNA expression levels of TNF death receptor pathway members, RNAse protection assays (RPAs) were performed. HEK-293 cells expressing the 91 kDa MEKK1 kinase domain were lysed and RNA was extracted. RPA analysis using the hApo3d panel was performed as described in the Materials and methods section. Cells transfected with 91 kDa MEKK1 show a 2.7-fold increase in Fas mRNA expression and a 1.6fold increase in DR4 mRNA levels when compared to cells expressing a vector control (Figure 2a). Western blot analysis reveal that transfection of 91 kDa MEKK1 increased Fas protein levels by twofold and DR4 protein levels by fourfold when compared to cells transfected with vector alone (Figure 2b). Caspase 8 mRNA levels increased threefold following 91 kDa MEKK1 overexpression but failed to show significant increase in protein levels. No increase in DR5 mRNA and protein levels following transfection of 91 kDa MEKK1 in cells was detected (Figure 2b). Therefore, MEKK1 leads to upregulation of the DR4 and Fas death receptors. Inhibition of PI-3 kinase potentiates MEKK1-induced apoptosis In order to further define the regulation of MEKK1induced apoptosis, PI-3 kinase activity was inhibited, and the ability of MEKK1 to promote apoptosis was examined. PI-3 kinase is important in cellular survival and has been found to activate AKT and mediate protection of cell from apoptosis. HEK-293 cells were treated with 200 nM of a PI-3 kinase inhibitor, wortmannin, for 12 h, and apoptosis was determined as described above. Expression of MEKK1 alone induced apoptosis in 39% of cells transfected. MEKK1 expression in the presence of wortmannin induced apoptosis in 52% of cells (Figure 3). Thus, by inhibition of endogenous PI-3 kinase, apoptosis induced by overexpression of MEKK1 was potentiated by 24%. This result suggests that even basal levels of PI-3 kinase activity can protect cells by negative regulation of MEKK1-induced apoptosis. AKT blocks MEKK1-induced apoptosis and caspase 3-like protease activation AKT is activated by PI-3 kinase and blocks genotoxininduced apoptosis (Khwaja, 1999). Since MEKK1 is involved in genotoxin induced apoptosis, the ability of AKT to block MEKK1-induced apoptosis was exam-

Figure 2 MEKK1 upregulates DR4 and Fas mRNA and protein levels. HEK-293 cells were transfected with control vector or a 91 kDa MEKK1 vector. Thirty-six hours following transfection, RPA analysis was carried out as described in Materials and methods. Fold increases in (a) DR4 and (b) Fas were quantitated and standard deviation performed. (c) Western blotting was performed with HEK-293 cells transfected with 91 kDa MEKK1 or vector control. Twenty-four hours post-transfection, cells were lysed as described and blotted for DR4, DR5, Fas and caspase 8. The level of expression was determined using a STORM phosphorimager using enhanced chemifluorescence (ECF). The expression was equalized for loading variations by re-probing with bactin

Figure 3 Inhibition of PI-3 kinase potentiates MEKK1-induced apoptosis. HEK-293 cells were transfected with a MEKK1-green fluorescent protein (GFP) vector or control GFP vector. Twentyfour hours after transfection, cells were serum starved for 12 h with DMSO or 200 nm Wortmannin. Cells were then fixed using 3.8% paraformaldehyde and quantified in a blinded manner for expression of GFP protein and apoptotic nuclei Oncogene


6652

ined. HEK-293 cells were transiently transfected with full length MEKK1 in the presence or absence of a constitutively activated myristoylated AKT (myrAKT), wild-type AKT (wt-AKT), p35, or vector alone and stained for protein expression using anti-MEKK1 and anti-AKT antibodies. p35 is a protein previously shown to inhibit MEKK1 cleavage and apoptosis and thus acts as a control in these experiments. Expression of myr-AKT and wt-AKT resulted in increased AKT kinase activity as determined by an in vitro AKT kinase assay (data not shown). Per cent apoptosis was quantified using a TdT-based TUNEL assay. The number of cells expressing MEKK1 and staining positively for TdT were then counted by fluorescence microscopy. At least 400 cells were counted for each condition in three separate experiments. Forty-two per cent of cells expressing MEKK1 with pCMV5 empty vector were apoptotic. In cells co-expressing MEKK1 with either myr-AKT or wt-AKT, however, the percentage of apoptotic cells was 8.5% and 7.6%, respectively (Figure 4a). These results reflect an 80% inhibition of MEKK1-induced apoptosis by AKT. As an internal control, cells co-expressing p35, an inhibitor of caspases, had 14.7% apoptosis (Figure 4a). Thus, AKT strongly inhibits MEKK1-induced apoptosis. AKT blocks apoptosis by multiple mechanisms, including activation anti-apoptotic proteins and by inhibition of pro-apoptotic proteins. To determine if AKT specifically blocks MEKK1-induced caspase activation, HEK-293 cells were transiently transfected with full length MEKK1, with or without the expression of myr-AKT and wt-AKT. Caspase activity was determined using a caspase 3 consensus substrate (DEVE-AFC), which, when cleaved, produces a fluorescent product quantified as described, in the Materials and methods section. Expression of MEKK1 alone caused a 2.1-fold increase in caspase 3-like protease activity above baseline. This fold increase was reduced to below basal levels by co-expression with myr-AKT (0.87-fold) and to basal level by coexpression with wt-AKT (1.07-fold) (Figure 4b). These results are consistent with the hypothesis that AKT blocks MEKK1-induced apoptosis by inhibiting caspase 3-like protease activation. AKT inhibits cleavage of endogenous MEKK1, which requires caspase 3 proteases Endogenous MEKK1 is cleaved by caspase 3-like proteases following treatment with genotoxic agents. HEK-293 cells stably expressing myr-AKT or vector alone were treated with etoposide (100 mM) or UV-C (40 J/m2). Following treatment, cells were lysed and assayed for full-length endogenous MEKK1 by Western blotting. Forty-eight hours following etoposide treatment, cleavage of endogenous MEKK1 was inhibited in the presence of a constitutively active myrAKT when compared to cells expressing vector alone (Figure 5a). Following UV irradiation, inhibition of cleavage of full-length MEKK1 was also seen in cells expressing myr-AKT and wt-AKT as compared with Oncogene

Figure 4 Expression of myr-AKT and wt-AKT inhibit MEKK1induced apoptosis and caspase activation. (a) Cells were analysed by fluorescence microscopy for expression of MEKK1 and for positive TdT staining. The percentage of cells with apoptotic nuclei as determined by TdT staining for each condition was quantified in a blinded manner. At least 400 cells were counted for each condition in three separate experiments. (b) HEK-293 cells expressing full-length MEKK1 along with myr-AKT, wt-AKT, or p35 were lysed. Caspase activity was determined by measurement of production of a fluorescent cleavage product of a caspase 3 consensus substrate (DEVE-AFC), as described in the Materials and methods section. Levels of caspase activation are expressed as fold difference in comparison with cells expressing pCMV5 vector alone

cells expressing pCMV5 or b-gal alone (Figure 5b). These results show that AKT overexpression blocks initial cleavage of endogenous MEKK1 to its pro-apoptotic 91 kDa kinase domain fragment. To further define the specific caspase involved in MEKK1 cleavage, we investigated primary mouse embryonic fibroblasts (MEFs) with homozygous deletion of caspase 3. MEF caspase 37/7 cells were treated with etoposide for 24 and 48 h, and Western blotting was performed to detect the presence of uncleaved, endogenous full-length MEKK1. As shown in Figure


6653

induced apoptosis by blocking activation of the JNK pathway, HEK-293 cells were transiently transfected with MEKK1 in parental cells or in cells stably expressing myr-AKT. The level of JNK activation was determined as described in the Materials and methods section. Transfection of MEKK1 into parental cells or cells expressing myr-AKT led to equivalent levels of JNK activation (Figure 6). Myr-AKT or vector alone did not independently activate JNK activity (Figure 6, data not shown). Thus, AKT does not block MEKK1-induced JNK activation. Discussion Our results indicate that death receptor activation is required for MEKK1-mediated apoptosis. Cleavage of MEKK1 by caspase-3 proteases precedes MEKK1induced death in response to chemotherapy treatment. The mechanism by which MEKK1 initiates apoptosis involves upregulation of DR4 and Fas death receptor levels. Expression of kinase inactive MEKK1 blocks the upregulation of DR4 following etoposide treatment (Gibson et al., 2000). MEKK1 activation also increases Fas ligand expression in Jurkat T cells, suggesting that MEKK1 regulation of death receptor activation is an important mechanism for induction of apoptosis in different cell types (Faris et al., 1998a,b). Our results

Figure 5 Cleavage of endogenous MEKK1 following etoposide or ultraviolet radiation in HEK-293 cells expressing AKT. HEK-293 cells were treated with 100 mM etoposide or ultraviolet irradiation (UV). Forty-eight hours later, the cells were lysed as described in Materials and methods section, and Western blots for endogenous MEKK1 were performed. (a) HEK-293 cells stably expressing empty vector or myr-AKT were treated with etoposide for 48 h and Western blots for MEKK1 were performed. The blots were stripped and reprobed with b-actin antibodies. (b) HEK-293 cells were transiently transfected with vector alone, wt-AKT, or myr-AKT and treated with 40 J/m2 UV irradiation. The cells were then lysed, and Western blotting for endogenous MEKK1 was performed. (c) Caspase 3 7/7 MEFs were treated with 100 mM etoposide or DMSO alone for 24 and 48 h, and Western blotting was performed for endogenous MEKK1 and b-actin

5c, cells lacking caspase 3 show no cleavage of fulllength MEKK1, while MEKK1 in wild-type control cells undergoes protease cleavage. Thus, cleavage of MEKK1 into its pro-apoptotic 91 kDa domain specifically requires caspase 3 proteases. Prevention of MEKK1-induced apoptosis by AKT is not mediated by inhibition of JNK activation MEKK1 is known to active c-Jun N-terminal kinase (JNK), a MAP kinase family member. JNK activation has been postulated to be involved with the induction of apoptosis. To determine if AKT blocks MEKK1-

Figure 6 JNK activation following expression of MEKK1 in HEK-293 cells. Parental or cells stably expressing myr-AKT were transfected with MEKK1 cDNA of empty vector and analysed for JNK activity in a GST-c-Jun assay. (a) Phosphorylated GST-c-Jun following JNK kinase assay as described in Materials and methods section was detected by phosphorimager. (b) Kinase activity was quantified by phosphorimaging analysis of the extent of phosphorylation of GST-c-Jun. Standard error was calculated from three independent experiments Oncogene


6654

indicate that MEKK1 activates death receptor apoptotic signaling pathways to induce apoptosis. Inhibition of an upstream regulator of AKT activation increases MEKK1-induced apoptosis. AKT blocks MEKK1-induced apoptosis caspase amplification by inhibiting cleavage of MEKK1 by caspase 3, thereby preventing release of the pro-apoptotic 91 kDa kinase fragment of MEKK1. This provides evidence that the apoptotic effects of MEKK1 are mediated by its cleavage into a 91 kDa kinase fragment. Correspondingly, it has been shown that caspase-3 is required for programmed cell death in fibroblasts in response to chemotherapy treatment and for apoptosis in ES cells following UV irradiation (Woo et al., 1998). The direct mechanism by which AKT blocks MEKK1 induced caspase activation is unknown, but our findings indicate that it is by inhibiting caspase 3 activity and thereby blocking subsequent MEKK1 cleavage to its 91 kDa kinase fragment. One mechanism by which caspase 3 activity is blocked is by the inhibitor of apoptosis (IAP) family of anti-apoptotic proteins. Cisplatin causes activation of caspase 3 and caspase 9 and is blocked by XIAP overexpression (Asselin et al., 2001). Further, the IAP family members XIAP and survivin are induced by VEGF in endothelial cells. VEGF is important in cell survival during angiogenesis and vasculogenesis in part due to AKT activation (Tran et al., 1999). AKT is also implicated in NF-kB modulation of XIAP levels, conferring resistance to apoptosis in macrophages (Lin et al., 2001). Thus, AKT may affect activation of proteins involved in caspase 3 inhibition, thereby blocking MEKK1’s ability to generate apoptotic signaling by upregulation of death receptors. Evidence suggests that death receptor activation plays a role in genotoxin-induced apoptosis. For example, increased expression of Fas ligand following doxorubicin treatment contributes to the induction of apoptosis (Friesen et al., 1996). Treatment with etoposide results in increased expression of DR4 and DR5 in both breast and lung cancer cells and is a proposed mechanism for genotoxin-induced apoptosis (Gibson et al., 2000). Alternatively, inhibition of TRAIL binding to DR4 and DR5 reduces the apoptotic response to etoposide (Gibson et al., 2000). Further, preliminary studies have shown the ability of TRAIL to reduce the size of human tumors in mice. In combination with genotoxic agents, TRAIL can eradicate some human tumors in mice and can lead to a synergistic apoptotic response in some breast cancer cell lines (Gibson et al., 2000; Griffith and Lynch, 1998; Gura, 1997). These responses could at least partially be explained by the upregulation of DR4 and DR5 expression. We have shown that AKT is effective at inhibition of MEKK1 and etoposideinduced apoptosis. Thus, blocking AKT or its upstream regulators could potentially result in enhancement of the apoptotic signal by TNF death receptor pathways, particularly cells with increased expression of AKT. Oncogene

Activation of MEKK1 leads to JNK activation. JNK has been implicated in the induction of apoptosis. In the presence of AKT, MEKK1 mediated JNK activation was not affected. This result suggests that JNK activation is not responsible for MEKK1’s proapoptotic effects and that AKT’s prevention of MEKK1-induced apoptosis is unrelated to MEKK1’s regulation of JNK. It is still possible, however, that AKT could block downstream events following JNK activation leading to inhibition of apoptosis. Since expression of kinase inactive MEKK1 fails to block JNK activation by etoposide (data not presented) but effectively blocks etoposide-induced apoptosis, it is unlikely that JNK is playing a major role in MEKK1induced apoptosis. Indeed, MEKK1 knock out fibroblasts show a decreased JNK response to cell stresses that alter the cytoskeletal structure and an increased apoptotic response to these stresses, suggesting that MEKK1-induced JNK activation may actually be protective (Yujiri et al., 1998). It is becoming more important to characterize the relationship between apoptotic and oncogenic signaling pathways in order to tailor more targeted therapies for cancer cells. Knowledge of the mechanisms by which cells regulate apoptosis can lead to potentiation of these pathways using specialized chemotherapies. Further, delineation of oncogenic pathways that subvert apoptotic pathways or effects of chemotherapeutic reagents will allow generation of more targeted cancer treatments to block these effects. Cumulatively, our results show that death receptor activation plays an important role in MEKK1-induced apoptosis and that AKT blocks initial MEKK1 cleavage and subsequent induction of caspase activation and apoptosis. Inhibiting the upstream activator of AKT, PI-3 kinase, potentiates MEKK1-induced apoptosis. Our findings identify potential molecular targets that may be effective at regulating TNF receptor and MEKK1-mediated apoptosis and overcoming survival signals such as AKT, leading to more targeted cancer therapies.

Materials and methods Reagents Dr Thomas Franke generously contributed AKT constructs. Constitutively active myristoylated c-AKT (myr-AKT) and wild type c-AKT (wt-AKT) were expressed in pCMV5. The MEKK1 constructs used have been previously described. HEK-293 cells were used for all transfections and for generation of AKT stable cell lines. AKT kinase assay HA-tagged myr-AKT was transfected by lipofectamine into HEK-293 cells and immunoprecipitated with anti-HA antibodies following serum starvation (0.1% BCS) for 12 h. The kinase assay was performed using a GSK-3 peptide substrate in the presence of g32P-ATP and quantified by scintillation counting.


6655

Apoptosis assay HEK-293 cells grown on glass coverslips were transfected with appropriate constructs. Following 0.1% serum starvation for 12 or 36 h, cells were fixed in 2% paraformaldehyde and permeabilized with 0.2% Triton X-100. After washing in PBS, cells were blocked with filtered cultured medium and incubated in TdT reaction mix (Boehringer). After washing, coverslips were incubated with the appropriate primary antibodies (rabbit anti-MEKK1, goat anti-AKT and Hoechst stain). The coverslips were then washed and incubated with secondary antibodies (Cy5 conjugated goat anti-rabbit, FITC conjugated donkey anti-goat, and Cy3 conjugated streptavidin). Images were collected using a Leica DMRXA microscope and analysed with SlideBook v2.0 software. Cells were quantified in a blinded manner. RNAse protection assay A RiboQuant Multi-Probe RNAse Protection Assay System (Pharmingen) was used according to the manufacturer’s instructions. A hAPO3c probe set containing DNA templates for caspase 8, FASL, Fas, DR3, DcR1, DR4, DR5, TRAIL, TNFR p55, TRADD, RIP, L32, and GAPDH (Pharmingen) was used for T7 RNA-polymerase direct synthesis of g32-PUTP-labeled anti-sense RNA probes. The probes were hybridized with 20 mg of RNA isolated from HEK-293 cells using RNAzol B (Tel-Test, Inc.). Samples were then digested with RNAse to remove single-stranded (non-hybridized) RNA. Remaining probes were resolved on denaturing polyacrylamide gels. Quantitation was made by phosphorimager analysis. Western blotting HEK-293 cells transfected with myr-AKT, wt-AKT, and the negative controls b-gal and pCMV5 were lysed in a TGH buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM HEPES, pH 7.3, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride, 10 mg/ml leupeptine, and 10 mg/ml aprotinin) following UV irradiation (40 J/cm2) or etoposide treatment. After rotation at 48C for 30 min, lysates were centrifuged at 14 000 g for 30 min, supernatants were collected, and protein concentration was determined by Bradford analysis. Generally, 0.5 mg of protein was separated by 10% SDS – PAGE, and transferred to a nitrocellulose membrane. Membranes were probed with MEKK1 specific antibodies generated in our lab or a C-22 MEKK1 antibody (Santa-Cruz). In addition, HEK-293 cells

were transfected with 91 kDa MEKK1 using Superfect reagent (Qiagen) and lysed as described above. Hundred mg of protein was loaded on a SDS page gel and Western blotted with antibodies against DR4 (Santa Cruz), DR5 (Santa Cruz), caspase 8 (Upstate Biotech.), and Fas (Santa Cruz). The blots were visualized using enhance chemiluminescence PLUS kit (Pharmacia) and analysed on a STORM phosphorimager (Molecular Dynamics) for fluorescence. The blots were stripped and re-probed with anti-b-actin (Sigma) to determine equal loading and analysed as described above. Caspase assay HEK-293 cells were co-transfected with 1.2 mg total DNA of full-length MEKK1 and myr-AKT, wt-AKT, p35, or pCMV5. Following serum starvation in 0.1% BCS, cells were lysed in 50 mM Tris (pH 7.4), 1 mM EDTA, and 10 mM digitonin for 10 min at 378C. Following a brief vortex, cell lysates were centrifuged at 14 000 g for 10 min. Sixty mg of lysate proteins were incubated with 5 mM of a DEVE-caspase substrate (Bachem). Generally, 0.5 mg lysates were used for Western blotting to confirm transfection levels. Fluorescence was then monitored with an excitation wavelength of 380 nm and emission wavelength of 460 nm to determine activity by a luminometer. Fluorescence of the substrate alone was subtracted in each case. c-Jun kinase assay c-Jun kinase (JNK) activity was measured using a solid phase kinase assay in which glutathione-S-transferase-c-Jun (1-79) (GST-Jun) bound to glutathione-Sepharose 4B beads was used to affinity purify JNK from transfected cell lysates. HEK-293 cells were lysed in 70 mM b-glycero-phosphate, 1 mM EGTA, 100 mM Na3VO4, 1 mM DTT, 2 mM MgCl2, 0.5% Triton X-100, and 20 mg/ml of aprotinin. Following centrifugation at 10 000 g for 5 min, cleared lysates were collected and protein concentration was normalized by the Bradford assay. JNK phosphorylation was quantified using a STORM phosphorimager.

Acknowledgements AH Bild was supported by a DOD predoctoral fellowship DAMD17-98-1-8297. SB Gibson was supported in part by Canadian Institutes of Health Research.

References Ashkenazi A and Dixit VM. (1999). Curr. Opin. Cell Biol., 11, 255 – 260. Asselin E, Mills GB and Tsang BK. (2001). Cancer Res., 61, 1862 – 1868. Blume-Jensen P and Hunter T. (2001). Nature, 411, 355 – 365. Cardone MH, Salvesen GS, Widmann C, Johnson G and Frisch SM. (1997). Cell, 90, 315 – 323. Datta SR, Brunet A and Greenberg ME. (1999). Genes Dev., 13, 2905 – 2927. Deak JC, Cross JV, Lewis M, Qian Y, Parrott LA, Distelhorst CW and Templeton DJ. (1998). Proc. Natl. Acad. Sci. USA, 95, 5595 – 5600.

Di Cristofano A, Kotsi P, Peng YF, Cordon-Cardo C, Elkon KB and Pandolfi PP. (1999). Science, 285, 2122 – 2125. Faris M, Kokot N, Latinis K, Kasibhatla S, Green DR, Koretzky GA and Nel A. (1998a). J. Immunol., 160, 134 – 144. Faris M, Latinis KM, Kempiak SJ, Koretzky GA and Nel A. (1998b). Mol. Cell. Biol., 18, 5414 – 5424. Friesen C, Herr I, Krammer PH and Debatin KM. (1996). Nat. Med., 2, 574 – 577. Gebauer G, Mirakhur B, Nguyen Q, Shore SK, Simpkins H and Dhanasekaran N. (2000). Int. J. Oncol., 16, 321 – 325. Gibson S, Widmann C and Johnson GL. (1999). J. Biol. Chem., 274, 10916 – 10922.

Oncogene


6656

Gibson SB, Oyer R, Spalding AC, Anderson SM and Johnson GL. (2000). Mol. Cell. Biol., 20, 205 – 212. Golstein P. (1997). Curr. Biol., 7, R750 – R753. Green DR. (2000). Cell, 102, 1 – 4. Griffith TS and Lynch DH. (1998). Curr. Opin. Immunol., 10, 559 – 563. Gura T. (1997). Science, 277, 768. Khwaja A. (1999). Nature, 401, 33 – 34. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ and Johnson GL. (1993). Science, 260, 315 – 319. Lee SH, Shin MS, Kim HS, Lee HK, Park WS, Kim SY, Lee JH, Han SY, Park JY, Oh RR, Kang CS, Kim KM, Jang JJ, Nam SW, Lee JY and Yoo NJ. (2001). Oncogene, 20, 399 – 403. Lin H, Chen C and Chen BD. (2001). Biochem. J., 353, 299 – 306. Mullauer L, Gruber P, Sebinger D, Buch J, Wohlfart S and Chott A. (2001). Mutat. Res., 488, 211 – 231. Pan G, O’Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J and Dixit VM. (1997). Science, 276, 111 – 113. Panka DJ, Mano T, Suhara T, Walsh K and Mier JW. (2001). J. Biol. Chem., 276, 6893 – 6896. Schlesinger TK, Fanger GR, Yujiri T and Johnson GL. (1998). Front Biosci., 3, D1181 – D1186. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P and Ashkenazi A. (1997). Science, 277, 818 – 821.

Oncogene

Shin MS, Kim HS, Lee SH, Park WS, Kim SY, Park JY, Lee JH, Lee SK, Lee SN, Jung SS, Han JY, Kim H, Lee JY and Yoo NJ. (2001). Cancer Res., 61, 4942 – 4946. Soh JW, Mao Y, Liu L, Thompson WJ, Pamukcu R and Weinstein IB. (2001). J. Biol. Chem., 276, 16406 – 16410. Straus SE, Jaffe ES, Puck JM, Dale JK, Elkon KB, RosenWolff A, Peters AM, Sneller MC, Hallahan CW, Wang J, Fischer RE, Jackson CM, Lin AY, Baumler C, Siegert E, Marx A, Vaishnaw AK, Grodzicky T, Fleisher TA and Lenardo MJ. (2001). Blood, 98, 194 – 200. Tran J, Rak J, Sheehan C, Saibil SD, LaCasse E, Korneluk RG and Kerbel RS. (1999). Biochem. Biophys. Res. Commun., 264, 781 – 788. Tschopp J, Irmler M and Thome M. (1998). Curr. Opin. Immunol., 10, 552 – 558. Widmann C, Gerwins P, Johnson NL, Jarpe MB and Johnson GL. (1998). Mol. Cell. Biol., 18, 2416 – 2429. Widmann C, Gibson S, Jarpe MB and Johnson GL. (1999). Physiol. Rev., 79, 143 – 180. Widmann C, Johnson NL, Gardner AM, Smith RJ and Johnson GL. (1997). Oncogene, 15, 2439 – 2447. Woo M, Hakem R, Soengas MS, Duncan GS, Shahinian A, Kagi D, Hakem A, McCurrach M, Khoo W, Kaufman SA, Senaldi G, Howard T, Lowe SW and Mak TW. (1998). Genes Dev., 12, 806 – 819. Yujiri T, Sather S, Fanger GR and Johnson GL. (1998). Science, 282, 1911 – 1914.