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Vol. 19, Issue 10, 4366-4373, October 2008

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Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

Xinxin Gao^*, and Jin Zhang^*,

*Department of Pharmacology and Molecular Sciences and The Solomon H. Snyder Department of Neuroscience and Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Submitted May 2, 2008; Revised July 31, 2008; Accepted August 6, 2008
Monitoring Editor: Sandra L. Schmid

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

Address correspondence to: Jin Zhang (jzhang32{at}jhmi.edu)

Abbreviations used: AktAR, Akt activity reporter; BKAR, B kinase activity reporter; FOXO, forkhead transcription factor O; FRET, fluorescence resonance energy transfer; IGF-1, insulin-like growth factor-1; InPAkt, indicator for 3' phosphoinositides based on Akt; MβCD, methyl-β-cyclodextrin; PDGF, platelet-derived growth factor; PH domain, pleckstrin homology domain; PI3K, phosphatidylinositol 3-kinase; PI(4,5)P₂, phosphatidylinositol (4,5)-bisphosphate; PI(3,4,5)P₃, phosphatidylinositol (3,4,5)-triphosphate.

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