Phosphatidylinositol 4,5-bisphosphate-specific AKT1 is oncogenic

Abstract

The protein kinase AKT1 (v-akt murine thymoma viral oncogene homolog 1), also referred to as protein kinase B (PKB), is an essential mediator of the phosphatidylinositol 3-kinase signaling pathway. Elevated activity of AKT1 is common in human cancer. Localization at the plasma membrane, leading to enhanced phosphorylation and activation of AKT1, is an important factor determining the oncogenicity of this kinase. Although the phosphatidylinositol 3-kinase signaling pathway is frequently upregulated in cancer, cancer-specific mutations in AKT1 are not common. Recently, such a mutation has been identified in breast, colon and ovarian cancers. The mutation is located in the pleckstrin homology (PH) domain of AKT1 and results in a glutamic acid to lysine substitution at residue 17. The resultant change in the conformation of the PH domain facilitates membrane binding of the mutant protein. Here we show that exchange of the PH domain leading to preferential binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) over phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) constitutively activates AKT1. AKT1 with this altered PIP affinity induces oncogenic transformation in cultures of chicken embryo fibroblasts and causes neoplastic growth and angiogenesis in the chorioallantoic membrane of the chicken embryo. Gain-of-function mutants of AKT1 may not be affected by PI3K inhibitors that are currently in development. Therefore, AKT1 remains a distinct and important cancer target.

Figure 1. PIP₂-specific AKT1 induces oncogenic transformation in CEF

(a) Schematic structure of AKT1 constructs. Human full-length AKT1 (NM_005163.2) was fused to an HA epitope tag on the carboxy terminus. E17K-AKT1 and myrAKT1 were generated using human wild-type AKT1 as template for PCR. The chimeric constructs GRP-AKT1 and PLC-AKT1 were obtained by substituting the PH domain of AKT1 with either the PH domain of GRP1 (NM_004227.3) or PLCδ1 (NM_006225). All constructs were subsequently cloned into the avian retroviral expression vector RCAS(A) . (b) Focus assay with AKT1 constructs. CEF were transfected with RCAS(A) encoding wild-type AKT1, E17K-AKT1, myrAKT1, GRP-AKT1 or PLC-AKT1 using the dimethyl sulfoxide/polybrene method . Cells were overlaid with agar medium every 2–3 days and stained with 2% (w/v) crystal violet in 10% (v/v) methanol after 17 days. A representative experiment out of three independent ones is shown.

Figure 1. PIP₂-specific AKT1 induces oncogenic transformation in CEF

(a) Schematic structure of AKT1 constructs. Human full-length AKT1 (NM_005163.2) was fused to an HA epitope tag on the carboxy terminus. E17K-AKT1 and myrAKT1 were generated using human wild-type AKT1 as template for PCR. The chimeric constructs GRP-AKT1 and PLC-AKT1 were obtained by substituting the PH domain of AKT1 with either the PH domain of GRP1 (NM_004227.3) or PLCδ1 (NM_006225). All constructs were subsequently cloned into the avian retroviral expression vector RCAS(A) . (b) Focus assay with AKT1 constructs. CEF were transfected with RCAS(A) encoding wild-type AKT1, E17K-AKT1, myrAKT1, GRP-AKT1 or PLC-AKT1 using the dimethyl sulfoxide/polybrene method . Cells were overlaid with agar medium every 2–3 days and stained with 2% (w/v) crystal violet in 10% (v/v) methanol after 17 days. A representative experiment out of three independent ones is shown.

Figure 2. Tumor induction and angiogenesis induced by PIP₂ affine AKT1 constructs in the CAM of the chicken embryo

CAM assays were performed with fertilized chicken eggs (White Leghorn) obtained from Charles Rivers Breeding Laboratories (Wilmington, MA). Eggs were incubated at 37.5 °C and 70% humidity for 9 days. Eggs were candled to confirm embryo growth and to locate vascularized areas of the CAM. Two holes were drilled, one into the air sac at the bottom of the eggs, another one at the lateral end above the blood vessels, penetrating through the outer shell membrane without injuring the CAM. Applying suction to the air sac exposed the CAM. CAMs were inoculated with AKT1, E17K-AKT1, myrAKT1, GRP-AKT1, PLC-AKT1 or empty vector. Eggs were sealed and incubated for another 9 days. A representative experiment out of three independent ones is shown.

Figure 3. Phosphorylation events associated with expression of Akt constructs

CEF transfected with RCAS constructs encoding wtAKT, E17K-AKT, myrAKT, GRP-AKT, PLC-AKT or empty vector were incubated under normal and serum starved condition. Whole cell lysates were electrophoresed and analysed by immunoblotting with anti-phospho-AKT (S473), anti-phospho-AKT (T308), anti-phospho-S6 (S235/S236), anti-phospho-GSK-3b (S9), anti-HA and anti-β-Actin, all obtained from Cell Signaling Technology® (Danvers, MA).

Figure 4. Intracellular localization of Akt constructs

CEF transfected with PLC-AKT1 (A–C) or GRP-AKT1 (D–F) constructs were incubated under serum starved conditions. Cells were fixed with formaldehyde and probed using an Anti-HA antibody. Images were taken at 100× maginification on an Axiovert 100TV fluorescence microscope.