TY - GEN
T1 - Illuminating the phosphatidylinositol 3-kinase/Akt pathway
AU - Ni, Qiang
AU - Fosbrink, Matthew
AU - Zhang, Jin
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
KW - Akt
KW - Fluorescence resonance energy transfer
KW - Fluorescent biosensor
KW - Fluorescent protein
KW - Live-cell imaging
KW - Phosphatidylinositol 3-kinase
KW - Phosphatidylinositol- 3,4,5-triphosphate
UR - http://www.scopus.com/inward/record.url?scp=42149102620&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=42149102620&partnerID=8YFLogxK
U2 - 10.1117/12.765524
DO - 10.1117/12.765524
M3 - Conference contribution
AN - SCOPUS:42149102620
SN - 9780819470430
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Small Animal Whole-Body Optical Imaging Based on Genetically Engineered Probes
T2 - Small Animal Whole-Body Optical Imaging Based on Genetically Engineered Probes
Y2 - 21 January 2008 through 22 January 2008
ER -