Alharbi, ZMS; (2015) Identification and characterization of novel mTOR splicing isoforms. Doctoral thesis , UCL (University College London).

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Abstract

mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase which belongs to the family of phosphoinositide 3-kinase related kinases (PIKK), which also includes ATR, ATM, DNA-PK, SMG1 and TRRAP. mTOR contains several conserved protein-protein interaction modules at the N-terminus and a protein kinase domain at the C-terminus. The regulatory interactions of mTOR are mainly mediated by HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, and TOR1) repeats and FAT (FRAP, ATM and TRRAP) domains. In contrast to other PIKK family members, mTOR possesses a unique FRB (FKBP12/Rapamycin Binding) domain which mediates the interaction with the FKBP12/rapamycin inhibitory complex. mTOR is a key component of two distinct multi-protein complexes in mammalian cells, termed mTOR complex 1 (TORC1) and mTOR complex 2 (mTORC2). A diverse range of extracellular and intracellular signals stimulate both mTOR complexes to regulate cell growth, survival and proliferation. Dysregulation of mTOR signalling has been implicated in various human pathologies, including cancer, inflammation, neurodegenerative and metabolic disorders. Therefore, mTOR is an attractive target for drug discovery. In cancer research, mTOR inhibitors have shown a potent anti-proliferative activity in pre-clinical studies and several of these are currently being evaluated in clinical trials. There is only one mTOR gene in higher vertebrates, which is known to encode two splicing isoforms: mTORα and mTORβ. In contrast to the full length mTORα protein, mTORβ lacks most of its protein-protein interaction modules, HEAT and FAT, but retains domains responsible for FKBP12/rapamycin binding, protein kinase activity and regulation. Importantly, mTORβ was shown to shorten considerably the G1 phase of the cell cycle, to stimulate cell proliferation and to possess oncogenic potential in cell-based and xenograft studies. Like other PIKK family members, there is increasing evidence of the presence of mTOR splicing isoforms. The main aim of this study was to identify new mTOR splicing isoforms and to determine their molecular characterization. In addition we aim to explore the ability of these isoforms to phosphorylate known mTOR targets such as 4E-BP1. Furthermore, we aim to study the role of the new isoforms in the mTOR–mediated cellular processes, such as cell proliferation, and the contribution of the identified isoforms in the oncogenic characteristics of cells. In this study, we have employed bioinformatics, biochemical, cell and molecular techniques to identify and characterize two novel mTOR splicing isoforms, denoted mTORδ and mTORγ. When compared to mTORα, the mTORδ splice variant contains only the N-terminal HEAT repeats and a unique C-terminal region, while the mTORγ isoform possesses a 12 amino acid deletion in the kinase domain. The existence of the mTORδ isoform was confirmed at mRNA and protein levels by identifying corresponding EST clones and detecting the splice variant with specific anti–mTORδ antibodies. Furthermore, we have found several EST clones corresponding to the mTORγ splicing variant. In contrast to mTORα and the mTOR activated mutant S2215Y, the stably expressed mTORδ and mTORγ lack the kinase activity in vitro. It was also found that stable overexpression of mTORδ and mTORγ splice variants in HEK293T cells inhibits cell proliferation and colony formation in soft agar. These findings suggest that identified novel isoforms have the potential to regulate the mTOR signalling pathway in a dominant–negative manner.

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