| Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that Bax and Bak are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are protected from NO-induced cell death. The individual loss of Bid, Bim, Puma, Bad and Noxa, or the combined loss of Bim and Puma with decreased Bid, does not prevent NO-induced cell death. Even though NO increases the binding of Bim to the anti-apoptotic protein Bcl-x L, the individual loss of Bim does not prevent NO-induced cell death. Proteomic analysis reveals novel protein interactions occur following treatment with NO. Specifically, myosin regulatory chain-2A, myosin light chain polypeptide 6 and calmodulin, all of which are components the myosin motor complex, bind to Bcl-xL following treatment with NO. We speculate that the myosin motor complex plays a role in the negation of Bcl-xL . Additionally, the anti-apoptotic protein Mcl-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO induces Bax/Bak-dependent cell death through the negation of pro-survival Bcl-2 proteins. Bcl-xL is negated in part by Bim and the myosin motor complex, and Mcl-1 is degraded through the ASK1-JNK1 pathway. |
