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Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being
developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often
dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such
biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid
second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation
process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined
nuclear phosphatidylinositol-3,4,5-triphosphate (PIP3) in living cells using nucleus-targeted InPAkt. Second, we
analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and
nucleus-targeted BKAR to simultaneously monitor PIP3 dynamics and Akt activity in the same cell. Of note, results from
these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the
nucleus in the presence of high levels of PIP3 at the plasma membrane. This finding has led to a further study of the
action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this
regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent
on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new
regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.
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