Akt Phosphorylates and Negatively Regulates Apoptosis Signal-Regulating Kinase 1

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells

“…Our study reveals that abrogation of Nox4-generated ROS induced inhibition of phosphorylation of AKT and subsequent suppression of AKT-mediated phosphorylation of ASK1 on Ser-83, and that the AKT phosphorylation-defective ASK1-Ser83Ala mutant alone can substitute for DPI and siNox4RNA in apoptosis induction. Because phosphorylation of AKT on Thr-103 activates its kinase activity (Alessi et al, 1996) and the activated AKT decreases the ASK1 kinase activity through phosphorylation of ASK1 on Ser-83 (Kim et al, 2001), it is most likely that inhibition of AKT phosphorylation by DPI and siNox4RNAs reduced its kinase activity, which in turn blocked AKT-catalysed phosphorylation of ASK1, resulting in ASK1-mediated cell death (see Figure 9). In this scheme, cell death events are accompanied by PARP degradation and JNK activation (Figure 6).…”

“…pcDNA3.0-hemagglutinin (HA)-tagged wt human ASK1 (pcDNA3.0-HA-ASK1) and the serine-83-to-alanine mutant of pcDNA3.0-HA-ASK1 (pcDNA3.0-HA-ASK1Ser83Ala) were previously described (Kim et al, 2001). pcDNA3.0 wt AKT1 and AKT1 Lys179Ala were previously described (Ogawara et al, 2002).…”

“…Because recent findings suggest that ASK1, a key player in stress-responsive apoptosis, is one of the protein substrates for AKT (Kim et al, 2001), this raises the possibility that DPI-induced suppression of phosphorylation of AKT inhibits AKTdependent phosphorylation of ASK1 on Ser-83. To test this possibility, lysates prepared from DPI-loaded PANC-1 cells were subjected to immunoblotting with antibodies to phospho-ASK1 antibodies, which specifically recognize ASK1 phosphorylated on Ser-83.…”

“…AKT/ASK1 signaling is involved in DPI/siNox4RNA-induced apoptosis As AKT phosphorylation of ASK1 on Ser-83 inactivates the apoptotic function of ASK1 (Kim et al, 2001), we examined whether the AKT-ASK1 signaling is involved in DPI-induced apoptosis in PANC-1 cells. For this purpose, the effect of ASK1Ser83Ala mutant on cell death was examined.…”

“…Apoptosis assay Cells were plated on coverslips and processed for DPI treatment or siNox4RNAs and ASK1Ser83Ala (Kim et al, 2001) transfection, as described above. Double-strand DNA breaks were then detected by the ApopTaqR Peroxidase in situ apoptosis detection kit (Chemicon, Temecula, CA, USA) according to the manufacturer's instructions.…”