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A novel conditional Akt ‘survival switch’ reversibly protects cells from apoptosis

Gene Therapy volume 9, pages 233–244 (2002)Cite this article

Abstract

The anti-apoptotic Akt kinase is commonly activated by survival factors following plasma membrane relocalization attributable to the interaction of its pleckstrin homology (PH) domain with phosphatidylinositol 3-kinase (PI3K)-generated PI3,4-P2 and PI3,4,5-P3. Once activated, Akt can prevent or delay apoptosis by phosphorylation-dependent inhibition or activation of multiple signaling molecules involved in apoptosis, such as BAD, caspase-9, GSK3, and NF-κB and forkhead family transcription factors. Here, we describe and characterize a novel, conditional Akt controlled by chemically induced dimerization (CID). In this approach, the Akt PH domain has been replaced with the rapamycin (and FK506)-binding domain, FKBP12, to make F3-ΔPH.Akt. To effect membrane recruitment, a myristoylated rapamycin-binding domain from FRAP/mTOR, called M-FRB, binds to lipid permeable rapamycin (and non-bioactive synthetic ‘rapalogs’), leading to reversible heterodimerization of M-FRB with FKBP-ΔPH.Akt. Like endogenous c-Akt, we show that the kinase activity of membrane-localized F3-ΔPH.Akt correlates strongly with phosphorylation at T308 and S473; however, unlike c-Akt, phosphorylation and activation of inducible Akt (iAkt) is largely PI3K independent. CID-mediated activation of iAkt results in phosphorylation of GSK3, and contributes to NF-κB activation in vivo in a dose-sensitive manner. Finally, in Jurkat T cells stably expressing iAkt, CID-induced Akt activation rescued cells from apoptosis triggered by multiple apoptotic stimuli, including staurosporine, anti-Fas antibodies, PI3K inhibitors and the DNA damaging agent, etoposide. This novel inducible Akt should be useful for identifying new Akt substrates and for reversibly protecting tissue from apoptosis due to ischemic injury or immunological attack.

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Acknowledgements

We thank Gerald R Crabtree and Tim Clackson for a critical reading of the manuscript and M. Neiditch, S-H Lee, and E Lowenthal for technical assistance. We thank ARIAD Pharmaceuticals for generously providing AP22783 and the FRB1 template DNA, P Tsichlis for plasmid pCMV6-HA-Akt, M Greenberg for plasmid pCMV-HA-AktK179M, and R Lloyd for pTPOV-3816. This work was supported by NIH grants R01-CA87569 and U01-CA84296.

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  1. Department of Immunology, Baylor College of Medicine, Houston, TX, USA

    B Li, SA Desai, RA MacCorkle-Chosnek, L Fan & DM Spencer

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Li, B., Desai, S., MacCorkle-Chosnek, R. et al. A novel conditional Akt ‘survival switch’ reversibly protects cells from apoptosis. Gene Ther 9, 233–244 (2002). https://doi.org/10.1038/sj.gt.3301641

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