Actually, MAPKKKs link a variety of extracellular stimuli to cytoplasmic and nuclear effectors by activating downstream MAPK pathways (21). and DR5/TRAIL-R2) results in receptor aggregation and recruitment of the FADD (Fas-associated death domain) adaptor proteins, which subsequently induce the formation of the death-inducing signaling complex (DISC) involved in the activation of the caspase 8 initiator (3,4). Activated caspase 8 cleaves Bid and/or caspase 3 and initiates the mitochondrial apoptotic pathway (intrinsic pathway) and/or the caspase cascade (extrinsic pathway), respectively, eventually leading to cell death (5). Many reports indicate that TRAIL kills a variety of tumor cell lines, while leaving normal cells viable, which suggests that this protein may function as a specific cancer therapeutic agent (6). Although TRAIL is regarded as a potential anticancer agent, a considerable proportion of cancer cells, especially some highly malignant tumors, are resistant to apoptosis induction by TRAIL, and some cancer cells that TAK-593 are initially sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance) (7). However, the exact mechanisms of acquired TRAIL resistance are still largely unknown. Recently, we reported that Src, c-Cbl, and PI3K are involved in the phosphorylation of Akt during TRAIL treatment, and these phosphorylations are related to TRAIL-induced acquired resistance (8,9). In addition to the non-apoptotic TRAIL signaling of Akt phosphorylation, TRAIL also induces the activation of MAPK kinase pathways in a caspase 8-dependent manner (10C12). However, the biological roles of JNK and p38 MAPK activations in TRAIL-induced signaling are uncertain (5). Moreover, multiple mechanisms by which JNK or p38 are activated by TRAIL have been reported. For example, Varfolmeev et al. (11) suggested that FADD, caspase-8, RIP1, and TRAF2 are recruited within the primary death-inducing signaling complex (DISC), leading to the stimulations of JNK and p38, while Liu et al. (13) concluded that MEKK1 could be activated via TRAF2 and RIP to activate JNK in the absence of apoptotic conditions. We demonstrated the occurrence of Mst1-mediated caspase 8-dependent activation of mitogen-activated TAK-593 protein kinases (MAPKs) TAK-593 during TRAIL incubation, but the upstream entities of the MAPKs remain unidentified. The MAPKs are a family of kinases that transduce external signals to the nucleus in order to decide the fate of the cell (14). Usually, conventional MAPKs consist of three family members, JNK, p38, and ERK, which are involved in different cellular processes, including inflammation, cell proliferation and differentiation, and apoptosis (5). Notwithstanding the TRAIL-induced ERK activation, which is mainly associated with an anti-apoptotic function, the functions of JNK and p38 activation in TRAIL-induced signaling are varied and can be controversial depending on the cell types and cellular contexts involved (15C20). However, the upstream molecules of the MAPKs have not yet been classified. Actually, MAPKKKs link a variety of extracellular stimuli to cytoplasmic and nuclear effectors by activating downstream MAPK pathways (21). MEKK1, ASK1, TAK1, and MLK2 are well-known MAP3 kinases (22). In this Rabbit Polyclonal to SHP-1 (phospho-Tyr564) study, we clearly demonstrate that MEKK1 and MEKK4 transmit TRAIL-induced signals to JNK or p38 MAPK in caspase 8-dependent manners. Materials and Methods Cell culture A human prostate adenocarcinoma cell line, DU-145, was cultured in Dulbeccos modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 26 mM sodium bicarbonate. The cells were maintained in a humidified environment containing 5% CO2 and air at 37 C. Reagents and antibodies Polyclonal anti-phospho-ERK, anti-ERK, anti-p38, monoclonal anti-phospho-p38, and anti-caspase-8 were purchased from Cell Signaling (Beverly, MA, USA), and anti-ACTIVE (phosphoT183 and phosphoY185) JNK was purchased from Promega (Madison, WI, USA). Polyclonal anti-JNK1 and 14-3-3 , , , and were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-MEKK1 was purchased from Assay Designs (Ann Arbor, MI, USA). Anti-ASK1 was purchased from Millipore (Billerica, MA, USA). Monoclonal anti-PARP was purchased from Biomol International, L.P. (Plymouth Meeting, PA, USA). Anti-actin antibody was purchased from ICN (Costa Mesa, CA, USA). Caspase-8 inhibitor (Z-IETD-FMK) was purchased from Calbiochem (San Diego, CA, USA). All other chemicals TAK-593 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein extracts.