Context-dependent expression of a conditionally-inducible form of active Akt.

Soyeon Park,Nikolai Kholodilov,Thomas F Franke,Pascaline Aimé,Lloyd A Greene,Oren Levy,Robert E Burke,Tatyana Kareva,Ferenc Gallyas

doi:10.1371/journal.pone.0197899

Abstract

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.

Highlights

The serine/threonine kinase Akt, known as protein kinase B (PKB), is a critical downstream effector of the PI3K signaling pathway [1,2,3,4,5]
The Akt(E40K) construct used here consists of the mouse Akt1 isoform with a glutamate to lysine substitution at amino acid 40, located within the pleckstrin homology (PH) domain. This substitution is analogous to the reported star mutation in the PH domain-containing Bruton tyrosine kinase and reported to enhance the binding specificity of Akt to phospholipids at the plasma membrane, leading to higher basal activity that is sufficient to replicate multiple biological effects associated with Akt activation [42,43,44]
There was a low, but detectable level of HA staining in cultures transfected with Destabilizing Domain (DD)-Akt (E40K) but untreated with TMP compared to cultures transfected with an empty vector, indicating that there is a basal level of DD-Akt(E40K) expression without TMP

Summary

Introduction

The serine/threonine kinase Akt, known as protein kinase B (PKB), is a critical downstream effector of the PI3K signaling pathway [1,2,3,4,5]. Canonical Akt activation begins when a trophic factor binds to a receptor tyrosine kinase and activates PI3K, which phosphorylates lipids at the plasma membrane to generate phosphatidylinositol 3,4 bisphosphate (PI3,4P2) and phosphatidylinositol 3,4,5 triphosphate (PIP3). These phosphorylated lipids exhibit high affinity for the Akt PH domain and lead to its recruitment to the inner surface of the plasma membrane. Membrane translocation of Akt and binding of its PH domain to phospholipids leads to a conformational change from a “PH-in” to a “PH-out” conformation, allowing access of upstream kinases to the kinase domain and phosphorylation at two sites: Thr308 within the catalytic domain by PDK1 and Ser473 within the C-terminal hydrophobic domain by mammalian target of rapamycin complex 2 (mTORC2) and other kinases [6]. The degree to which the two canonical phosphorylation sites have distinct functions has not been fully determined; it has been reported that while phosphorylation of both sites is required for full catalytic activity, phosphorylation of the Thr308 site stimulates enzymatic activity by an estimated 100-fold, whereas phosphorylation of the Ser473 site can induce activity levels by an additional 7 to 10-fold [7,8,9,10,11]

Methods

Results

Conclusion