A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis.
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-P(2) and PI3,4,5-P(3). 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-kappaB 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-DeltaPH.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-DeltaPH.Akt. Like endogenous c-Akt, we show that the kinase activity of membrane-localized F3-DeltaPH.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-kappaB 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.
Li
B
B
Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
Desai
S A
SA
MacCorkle-Chosnek
R A
RA
Fan
L
L
Spencer
D M
DM
D017209:Apoptosis / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019169:Jurkat Cells; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000235:genetics; D020123:Sirolimus / Q000378:metabolism
D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; C494918:AKT1 protein, human