Lee). Abbreviations used in this paper TRAILtumor necrosis factor-related apoptosis-inducing ligandPAGEpolyacrylamide gel electrophoresisPARPpoly(ADP-ribose) polymeraseMAPKmitogen-activated protein kinaseJNKc-Jun, NH2-terminal kinaseERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinaseMEKK1MEK kinase 1ASK1apoptosis signal-regulating kinase 1SEK1stress-activated protein kinase/extracellular-signal regulated kinaseMLKmixed lineage kinaseTAK1TGF- activated-kinaseROSreactive oxygen speciesGCKgerminal center kinaseTRAF2TNF receptor-associated factor 2Mst1mammalian sterile 20-like kinase 1. signal to JNK/p38 in a caspase 8-dependent manner. 2.1-U6 hygro vector (Ambion, Inc., Austin, TX) was used for clonal cell lines. The inserts for hairpin siRNA into pwere prepared by annealing two oligonucleotides. For human SEK1 siRNA, the top strand sequence was 5_-GATCCACGCAAAGCACTGAAGTTGTTCAAGAGACAACTTCAGTGCTTTGCGTTTTTTTGGAAA-3, and the bottom strand sequence was 5-AGCTTTTCCAAAAAAACGCAAAGCACTGAAGTTGTCTCTTGAACAACTTCAGTGCTTTGCGTG-3. The annealed insert was Nodakenin cloned into p2.1-U6 hygro digested with BamHI and HindIII. The correct structure of p2.1-U6 hygro-SEK1 was confirmed by nucleotide Nodakenin sequencing. The resultant plasmid, pimmunocomplex kinase assay. As expected based upon our previous experience (12), increased MEKK1 activity by TRAIL was repressed by caspase 8 activation (Fig. 2A), and SEK1 mediated the MEKK1 signaling cascade to JNK during TRAIL treatment (Fig. 2B). Open in a separate window Open in a separate window Figure 2 Caspase 8-dependent MEKK1 activation during TRAIL treatment. (A) DU-145 cells were pretreated with caspase-8 inhibitor (Z-IETD-FMK 20 M, 30 min), followed by TRAIL treatment (200 ng/ml) for 2 or 4 h, and were lysed. Cell lysates were immunoprecipitated with anti-MEKK1 antibody. MEKK1 catalytic activity was determined by incubation with GST-SEK1 followed by GST-JNK1 as sequential substrates (upper panels). Phosphorylated-JNK, JNK1, or MEKK1 were detected with anti-ACTIVE JNK, anti-JNK1, or anti-MEKK1 antibodies, respectively. Cell lysates (lower panel) were immunoblotted with anti-PARP, anti-caspase-8, or anti-actin antibody, respectively. (B) Phosphorylated JNK in control vectorCtransfected (si-neg) or pSilencersi SEK1 stably transfected (si-SEK1) single cell clones was determined during TRAIL treatment from DU-145 cells. Lysates containing equal amounts of protein (20 g) were separated by SDS-PAGE and were immunoblotted with anti-ACTIVE JNK, anti-JNK1, anti-SEK1 and anti-actin antibody, respectively. C: control; T: TRAIL 14-3-3 interacts with MEKK1, and its dissociation from MEKK1 by TRAIL treatment is caspase 8-dependent As mentioned by Widmann et al. (24), full-size MEKK1 activated JNK independent of its truncated form (91 kDa) (Figs. 2A and 2B); 91 kDa of MEKK1 was only observed when TRAIL was treated to cells that Nodakenin overexpressed MEKK1 (data not shown). These results do not clearly elucidate the underlying mechanisms of MEKK1 activation. Widmann et al. (24) suggested that 14-3-3 proteins, which are a family of serine/threonine binding proteins that are expressed ubiquitously (25), have anti-apoptotic functions exerted by directly sequestering pro-apoptotic proteins, such as Bad (26,27). We examined the same possibility of sequestration of MEKK1 activity by 14-3-3 and release of MEKK1 from 14-3-3. First, we examined whether various 14-3-3 isotypes are cleaved by TRAIL treatment and found that there was no cleavage of 14-3-3 (Fig. 3A). Then, we investigated the associations of MEKK1 and various 14-3-3 isotypes. As shown in figure 3B, only 14-3-3 showed a meaningful change with MEKK1 during TRAIL treatment. In DU-145 cells, the dissociation of MEKK1 from 14-3-3 was inhibited by caspase 8 inhibitor (Fig. 3C), implying that caspase 8 activity is necessary for 14-3-3 release and subsequent MEKK1 phosphorylation and activation. Open in a separate window Open in a separate window Open in Nodakenin a separate window Figure 3 14-3-3 isotypes and MEKK1 during TRAIL treatment. (A) Various 14-3-3 isotypes were examined for cleavage by TRAIL (200 ng/ml) after various periods of time. (B) DU-145 cells were treated with TRAIL (200 ng/ml) for 2 h and lysed. Cell lysates were immunoprecipitated with anti-14-3-3 (left panel) or 14-3-3 (right panel) and immunoblotted with anti-14-3-3 / antibody or anti-MEKK1 antibody (upper panels). Cell lysates (lower panel) were immunoblotted with MEKK1 antibody. (C) DU-145 cells were pretreated with caspase-8 inhibitor (Z-IETD-FMK 20 M, 30 min), followed by TRAIL treatment (200 ng/ml) for 2 h, and were lysed. Cell lysates were immunoprecipitated with anti-14-3-3 antibody and immunoblotted with anti-MEKK1 antibody (upper panels). Cell lysates (lower panel) were immunoblotted with MEKK1 antibody, anti-PARP antibody, caspase-8 antibody, or anti-actin antibody, respectively. MEKK4 is responsible for p38 activation as well as JNK activation in TRAIL Rabbit Polyclonal to HSP90A treatment From the report indicating that MEKK4 can stimulate p38 as well as JNK activity (28), MEKK4 involvement of p38 activation during TRAIL treatment was examined using siRNA of MEKK4. As shown in figure 4A, p38 and JNK phosphorylation were repressed during TRAIL treatment when MEKK4 expression was downregulated.. Additionally, MEKK4 catalytic activity was also affected by caspase 8 activation (Fig. 4B). Open in a separate window Open in a separate window Figure 4 Involvement of MEKK4 in MAPKs phosphorylation during TRAIL treatment. (A) MAPKs phosphorylation induced by TRAIL treatment (200 ng/ml, 4 h) was examined after transfection of control siRNA or MEKK1 siRNA (left) or MEKK4 siRNA Nodakenin (right) into DU-145 cells. (B) DU-145 cells were pretreated with caspase-8 inhibitor (Z-IETD-FMK 20 M, 30 min), followed.