BACR9
The pro-apoptotic function of p38MAPK is regulated by its ASK1-dependent sustained phosphorylation
Danielle White, Sue Burchill
Leeds Institute of Molecular Medicine, Leeds, UK
Background
Sustained phosphorylation of p38MAPK initiates cell death in ESFT cells (Williamson et al, 2004, J Biol. Chem. 279: 47912-28; Myatt et al. 2005, Clin. Cancer Res. 11: 3136-48). We have examined the hypothesis that the generation of ROS and activation of the redox-controlled kinase ASK1 regulates the phosphorylation of p38MAPK and its pro-apoptotic function.
Method
ROS were measured using CM-H2DCFDA and flow cytometry. Phosphorylation of p38MAPK and its regulatory kinases MKK3/6 was measured by immunoblot. Phosphorylation of p38MAPK isoforms was quantified by ELISA and ASK1 activity by immune-complex kinase assay. ASK1-thioredoxin complex was detected by immunoprecipitation and immunoblot. The roles of ASK1 and p38MAPK were verified using siRNA, and of ROS using the anti-oxidant vitamin C. The effect of fenretinide on viable cell number was determined using the trypan blue exclusion assay.
Results
Fenretinide (3μM) increases the level of intracellular ROS, inducing oxidation of thioredoxin, release of ASK1 and a rapid increase in its kinase activity (5min). This preceded MKK3/6 and p38MAPK phosphorylation (10min); phosphorylation of p38MAPK was sustained >2h. p38MAPKα appears to be pro-apoptotic, but not δ or γ. Pre-treatment of cells with vitamin c inhibited fenretinide-induced increases in ROS and release of ASK1 from thioredoxin. Knock-down of ASK1 using siRNA prevented fenretinide-induced phosphorylation of MKK3/6 and p38MAPK, and was associated with rescue of ESFT cells from cell death. In contrast the cell survival effect of stem cell factor (20ng/ml) was associated with transient (10min) phosphorylation of p38MAPK.
Conclusion
ASK-1 dependent regulation of p38MAPK determines ESFT cell fate.
