Delivery and differentially phosphorylated AKT1 regulation in mammalian cells

Tarana Siddika, Western University

Abstract

Protein kinase B (AKT) is a serine and threonine kinase that performs a critical role in cell proliferation, growth, apoptosis, metabolism, and cell signalling, and faulty AKT activity contributes to various diseases. Phosphorylation at two regulatory sites, Thr308 and Ser473, activates AKT1, leading to phosphorylation of AKT1 targets. Thr308 and Ser473 reside in the catalytic and hydrophobic motif domain of AKT1, respectively. Ser473 is structurally similar to Thr308, but its side chain is smaller. These two sites have both distinct and overlapping regulatory effects on AKT1. The specific role of each AKT1 phosphorylation site in the regulation of downstream substrates, cell proliferation, and metabolism remained elusive. To characterize the individual roles of AKT1 phosphorylation sites in human cells, we fused site-specifically phosphorylated AKT1 with a cell-penetrating peptide, the trans-activator of transcription (TAT). By combining genetic code expansion with or without co-expression of the upstream kinase, I produced specific phospho-forms of TAT-tagged AKT1, including TAT-pAKT1^T308, TAT-pAKT1^S473 and TAT-ppAKT1^T308,S473. We found that TAT-AKT1 proteins retained enzymatic activity, and the TAT-tag effectively delivered the AKT1 proteins into HEK 293T cells without causing cytotoxicity. We confirmed the entry and efficiency of TAT-mCherry-AKT1 transduction by fluorescence microscopy. In cell-based experiments, I found that Thr308 phosphorylation leads to increased phosphorylation of the AKT1 target GSK-3α but not GSK-3β. Cells transduced with TAT-pAKT1^S473 and TAT-ppAKT1^T308,S473 stimulated the phosphorylation of both GSKα and GSKβ. All AKT1 phospho-forms stimulated downstream signalling to ribosomal protein S6 and enhanced cell proliferation to varying degrees. In epithelial ovarian cancer cells, I found that TAT-AKT1 entered the cells more efficiently compared to plasmid-based transfection. Using phopshoproteomics, I identified novel pathways and downstream substrates of pAKT1^T308 in HEK 293T cells. Finally, I used metabolomic analysis to uncover the role of each AKT1 phospho-form in glucose metabolism and mitochondrial regulation. In summary, the fusion of phosphorylated AKT1 variants with a cell-penetrating peptide enabled the characterization of phosphorylation-dependent substrate regulation, identification of new AKT1 substrates, and analysis of the role of differentially phosphorylated AKT1 variants in metabolism and oxidative phosphorylation.