Abstract 14687: Cardioprotective Signals Converge on Ribosomal Protein S6 to Reduce Cell Death
Objective The aim of this study is to get new insights into the mechanisms by which phosphatidylinositol 3-kinase (PI3K)/Akt signaling is connected to suppression of cell death.
Methods and Results Using an antibody that detects phosphorylation of serine and/or threonine on Akt consensus sequences (RXRXXS/T), we found that a 32kDa band was strongly phosphorylated by cardioprotective interventions such as ischemic preconditioning (IPC), diazoxide and insulin in perfused mouse hearts. Further analysis showed that the 32kDa protein was ribosomal protein S6 (Rps6). Rps6 phosphorylation at ser235/236 by IPC was inhibited by wortmannin, a PI3K inhibitor and PP242, a mammalian Target of Rapamycin complex (mTORC) inhibitor. On the other hand, diazoxide-induced Rps6 phosphorylation was blocked by chelerythrine, a PKC inhibitor. Furthermore, both 8-Br-cAMP, a PKA activator, and 8-CPT-cGMP, a PKG activator, also phosphorylated Rps6 at ser235/236. These findings demonstrate that multiple cytoprotective kinases can phosphorylate Rps6. Interestingly, Rps6 interacted with components of mTORC2 such as mTOR itself and rictor. Rps6-mTORC2 complex phosphorylated recombinant Akt at ser473 in vitro, which was enhanced by pretreatment with insulin. In HEK293 cells and neonatal cardiomyocytes, insulin- and isoproterenol-induced Akt/GSK3β signaling were attenuated by siRNA-mediated knockdown of Rps6 or pharmacological inhibition of mTOR by PP242. In isolated, buffer-perfused mouse hearts, IPC-induced Akt ser473 phosphorylation was also blocked by PP242. Finally, perfused mouse hearts were subjected to 20 min ischemia/120 min reperfusion. IPC reduced infarct size as a percentage of ventricular area from 53.7±3.8% in control hearts to 24.3±3.1%. This cardioprotective effect of IPC was completely blocked by wortmannin (47.4±1.6%) and attenuated by PP242 (40.4±2.7%).
Conclusion Rps6 phosphorylation is a key element of pro-survival signaling. Although Rps6 is known as a regulator of protein translation, Rps6 phosphorylation is a step in the PI3K signaling pathway leading to mTORC2 activation and serves as a positive feedback loop in cytoprotective signaling.
- © 2012 by American Heart Association, Inc.