Wang Ruiwen, Brattain Michael G
Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Cell Signal. 2006 Oct;18(10):1722-31. doi: 10.1016/j.cellsig.2006.01.020. Epub 2006 Feb 28.
To investigate issues about AKT/PKB nuclear localization in cells, we examined endogenous or transiently transfected AKT localization in cancer cell lines by immunofluorescence. We found that AKT can be detected in both the nucleus and cytoplasm of HEK 293, HeLa and MCF7E cells. It was found that an active process mediates AKT nuclear translocation as shown by fusing AKT with GFP3 protein. 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 segment does not have any apparent nuclear transport activity. These results implied that the signal mediating AKT nuclear translocation is conformation dependent, or more likely, is dependent upon association with other proteins. It was also found that AKT does not contain any apparent nuclear export signal. Furthermore, we found that nuclear AKT was activated in MCF7E cells upon stimulation. The possibility that nuclear activated AKT was translocated from the cytoplasm was excluded through the generation of a chimeric AKT protein, in which a strong nuclear localization signal was fused to the C-terminal of AKT.
为了研究细胞中AKT/PKB核定位的相关问题，我们通过免疫荧光检测了癌细胞系中内源性或瞬时转染的AKT定位。我们发现，在HEK 293、HeLa和MCF7E细胞的细胞核和细胞质中均能检测到AKT。通过将AKT与GFP3蛋白融合发现，存在一个活跃过程介导AKT核转位。GFP3-HA-AKT系列缺失突变体的细胞分布模式表明，AKT核转位需要AKT的多个片段，而单个片段没有任何明显的核转运活性。这些结果表明，介导AKT核转位的信号依赖于构象，或者更可能依赖于与其他蛋白质的结合。还发现AKT不包含任何明显的核输出信号。此外，我们发现MCF7E细胞在受到刺激后，细胞核中的AKT被激活。通过构建一种嵌合AKT蛋白（其中一个强核定位信号融合到AKT的C末端），排除了核激活的AKT从细胞质转位的可能性。
