ATP‐binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria‐derived activator of caspase and apoptosis in C2C12 cells

Abstract

Hydrogen peroxide (H(2)O(2)) is a well known oxidative stress inducer causing apoptosis of many cells. Previously, we have shown that heat shock pretreatment blocked the release of the second mitochondria-derived activator of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myoblast cells in response to H(2)O(2). The present study aimed to elucidate the underlying mechanism by over-expressing a major stress-inducible protein, heat shock protein (HSP) 70, and characterizing the resulting cellular changes. We demonstrate that HSP70 over-expression markedly inhibited the release of Smac and prevented the activation of caspases-9 and -3 and apoptosis in C2C12 cells under H(2)O(2) treatment. However, no direct interaction between HSP70 and Smac was observed by co-immunoprecipitation. Mutational analysis demonstrated that the ATP-binding domain of HSP70, rather than the peptide-binding domain, was essential for these observed HSP functions. Taken together, our results provide evidence supporting the role of HSP70 in the protection of C2C12 cells from H(2)O(2)-induced and Smac-promoted apoptosis by preventing the release of Smac from mitochondria, thereby inhibiting activation of caspases-9 and -3. This mechanism of HSP70 action is dependent on its ATP-binding domain but independent of its interaction with Smac protein.