The role of nuclear AKT in cell survival
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The overall objective of this dissertation is to investigate the mechanism of AKT nuclear translocation and the potential role of nuclear AKT in cell survival. We demonstrated that both endogenous and transiently transfected AKT could be transported into the nucleus of HEK293, HeLa and MCF7E cells. It was further shown that the nuclear translocation of AKT was dependent upon an active process as shown by fusing AKT with the GFP3 protein, which would be too large (∼150kDa) to diffuse through the nuclear pore complex. The cellular distribution pattern of serial deletion mutants from GFP3-HA-AKT revealed that more than one segment of AKT is required for AKT nuclear translocation, while the individual segments did not have any apparent nuclear transport activity. These results suggested that the signal responsible for AKT active nuclear translocation is possibly conformation dependent, or more likely, AKT does not contain a nuclear localization signal per se and is translocated into the nucleus through association with other nuclear proteins. In addition, it was found that AKT does not contain any apparent nuclear export signal. Classical AKT activation involves its phosphorylation at 308 and 473 sites directed by PH domain mediated binding to the cell membrane. The fact that the enzymes that are responsible for AKT phosphorylation (activation) could all be identified in the nucleus raised the possibility that nuclear localized AKT can be activated too. In this study, we found that nuclear AKT was activated in MCF7E cells upon stimulation. The possibility that nuclear activated AKT was translocated from the cytoplasm was further excluded through the generation of a chimeric AKT protein, in which a strong nuclear localization signal was fused to the C-terminal of AKT (HA-AKT-cNLS). Interestingly, it was found that the phosphorylation of AKT at the Ser473 site was severely impaired in HA-AKT-cNLS when compared to that of HA-AKT. This suggested that the maintenance of the C-terminal hydrophobic region of AKT is important for its phosphorylation at the Ser473 site. By specifically blocking endogenous cytoplasmic and nuclear AKT activity, we further demonstrated that both the cytoplasmic and nuclear AKT play a role in protecting cells against cisplatin-induced apoptosis, thus proving that nuclear AKT makes an important contribution to the function of AKT in cell survival.