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J. Biol. Chem., Vol. 282, Issue 50, 36634-36641, December 14, 2007

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Live-cell Molecular Analysis of Akt Activation Reveals Roles for Activation Loop Phosphorylation^*

Bharath Ananthanarayanan, Matthew Fosbrink, Meghdad Rahdar, and Jin Zhang¹

From the 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, Maryland 21205

Activation of the serine/threonine protein kinase Akt/PKB is a multi-step process involving membrane recruitment, phosphorylation, and membrane detachment. To investigate this process in the cellular context, we employed a live-cell fluorescence imaging approach to examine conformational changes of Akt and its membrane association. A fluorescence resonance energy transfer-based reporter of Akt action (ReAktion) reveals a conformational change that is critically dependent on the existence of a phosphorylatable threonine 308 in the activation loop, because mutations to either aspartate or alanine abolished the change. Furthermore, a mutant carrying a phosphorylation mimic at this position showed diminished membrane association, suggesting that this phosphorylation plays an important role of promoting the dissociation of activated Akt from the membrane. In addition, the membrane-associating pleckstrin homology domain was found to associate with the catalytic domain when Thr³⁰⁸ is phosphorylated, suggesting such an interdomain interaction as a mechanism by which phosphorylation within the catalytic domain can affect membrane association. These studies uncover new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

Received for publication, July 30, 2007 , and in revised form, October 9, 2007.

^* This work was supported by National Institutes of Health Grant CA122673, a Scientist Development Award from the American Heart Association, the Young Clinical Scientist Award Program of the Flight Attendant Medical Research Institute, 3M (to J. Z.); and the W. M. Keck Center (to B. A. and J. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 725 N. Wolfe St., Hunterian #307, Baltimore, MD 21205. Tel.: 410-502-0173; Fax: 410-955-3023; E-mail: jzhang32{at}jhmi.edu.

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