Cell Structure and Function
Online ISSN : 1347-3700
Print ISSN : 0386-7196
ISSN-L : 0386-7196

この記事には本公開記事があります。本公開記事を参照してください。
引用する場合も本公開記事を引用してください。

Development of a FRET biosensor with high specificity for Akt
Haruko MiuraMichiyuki MatsudaKazuhiro Aoki
著者情報
キーワード: Akt, FRET, EGF
ジャーナル フリー 早期公開

論文ID: 13018

この記事には本公開記事があります。
詳細
抄録

The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signaling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.

著者関連情報
© 2013 by Japan Society for Cell Biology
feedback
Top