Thesis Format

Integrated Article

Delivery and differentially phosphorylated AKT1 regulation in mammalian cells

Author

Tarana Siddika, Western UniversityFollow

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

O' Donoghue, Patrick

2nd Supervisor

Heinemann, Ilka

Co-Supervisor

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.

Summary for Lay Audience

Every year, many people suffer from diabetes, cardiac, and neurological disorders, as well as cancer, which is not only costly to treatment but also significantly impacts the lives of those diagnosed. In cancer, cells grow and divide out of control because certain essential proteins in the body start behaving incorrectly. For example, AKT1 protein is involved in many crucial cellular processes, such as cell growth and division, which helps to maintain normal cellular regulation of the body and avoid diseases. AKT1 becomes activated in cells when a phosphate group is added to two specific sites in the AKT1 protein: Threonine at position 308 and Serine at position 473. We can measure the activity of AKT1 by looking at its phosphorylation level; interestingly, this can sometimes be a sign of cancer. Although we know when AKT1 is activated, it adds phosphate groups to other proteins and regulates their functions. We do not fully understand how it acts when it is phosphorylated only at Threonine308 or Serine473 separately compared to when it is phosphorylated at both positions. To solve this, we developed a new way to study the different phosphorylated forms of AKT1. We attached the different forms of AKT1 to a particular protein called TAT, which helps AKT1 easily enter cells. We produced three versions of the TAT-tagged AKT1 protein: single phosphorylated forms, TAT-pAKT1^T308 and TAT-pAKT1^S473, and double phosphorylated TAT-ppAKT1^T308,S473. We found that all forms of AKT1 proteins made cells grow and divide more, but each one had different effects on the proteins. Later, TAT-AKT1 protein was delivered into ovarian cancer cells where we found that the TAT-tagged protein had better efficiency in entering inside the cells compared to other techniques. Finally, we looked at how different phosphorylation states of AKT1 affected glucose breakdown and energy production. When overactive, AKT1 can lead to many cancers, making this protein a key target for cancer treatments. The development of drugs to reduce the activity of AKT1 is rapidly expanding; through a better understanding of how AKT1 works and regulates other components in our cells, researchers hope to develop better treatments for diseases.

Recommended Citation

Siddika, Tarana, "Delivery and differentially phosphorylated AKT1 regulation in mammalian cells" (2024). Electronic Thesis and Dissertation Repository. 10177.
https://ir.lib.uwo.ca/etd/10177

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.