Delivery of Active AKT1 to Human Cells

Abstract

Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Thr308, Ser473), yet cell stimulation also activates many other kinases. To produce cells with specific AKT1 activity, we developed a novel system to deliver active AKT1 to human cells. We recently established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. Here, we fused AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) protein. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308 induced selective phosphorylation of the known AKT1 substrate GSK-3α, but not GSK-3β, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Ser240/244. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on AKT1 activity.

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“…AKT1 is a downstream effector of PI3K, becoming activated by localizing to the membrane after inositol-3,4,5-triphosphate (PIP3) phosphorylation by PI3K. The pleckstrin homology domain of AKT1 binds to PIP3, exposing the activation sites of AKT1 for phosphorylation of serine 473 and threonine 308 ( 77 ). The process is often implicated in proliferation, survival and angiogenesis pathways recurrently activated in numerous cancers, including breast cancer ( 78 ).…”

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“…GMP kinases and protein kinase C) and comprises of triple conserved domains: an N-terminal pleckstrin homology domain, a central kinase catalytic domain and a C-terminal extension motif (Siddika et al, 2022). AKT family proteins are encoded by three genes, including AKT1, AKT2, and AKT3.…”

“…The PI3K/AKT signal pathway is critical in various mammalian cell types, that controls cell proliferation, death, protein production, and so on (Figure 1) (He, Sun, et al, 2021). As the central molecules, PI3K protein is a heterodimeric protein, comprising P85 and P110 subunits (Bilanges et al, 2019) and AKT (also known as protein kinase B or PKB) which is a member of the AGC kinase family (related to AMP/GMP kinases and protein kinase C) and comprises of triple conserved domains: an N‐terminal pleckstrin homology domain, a central kinase catalytic domain and a C‐terminal extension motif (Siddika et al, 2022). AKT family proteins are encoded by three genes, including AKT1 , AKT2 , and AKT3 .…”