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Role of eIF3d on the mTOR signalling pathway
[Thesis]. Manchester, UK: The University of Manchester; 2016.
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Abstract
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family and participates in sensing and integration of a variety of environmental cues. As a central regulator of cell metabolism, mTOR has gained attention due to its role in many human diseases, including cancer and diabetes. Thus, great efforts are being made to understand signalling via the mTOR pathway as it represents an interesting pharmacological target. In higher eukaryotes, mTOR exists in two evolutionary conserved multi-protein complexes, TORC1 and TORC2. These complexes contain core components that define each complex and regulate their activities. mTORC1 signalling has been extensively studied but mTORC2 is still poorly understood. The discovery that mTORC2 is a key regulator of the activities of the AGC family kinases AKT, PKC and SGK1, and is involved in cellular processes such as proliferation and cell survival in response to growth factors, has stimulated the study of its activation and regulation. A core component of mTORC2 is stress-activated protein kinase interacting protein (SIN1), which is essential for the assembly and kinase activity of the complex and has been proposed to act as a scaffold to recruit mTOR substrates to facilitate their phosphorylation. A yeast two-hybrid screen uncovered novel SIN1 binding proteins that may be potential regulators of mTORC2 activity. One of these is subunit D of the Eukaryotic translation initiation factor 3 (eIF3d), a conserved protein involved in protein metabolism and cytoskeletal organization. This interaction has been confirmed by pull-down assays with overexpressed proteins in HEK-293 cells and the C-terminal region of eIF3d has been identified as critical for this interaction. Moreover, it has been confirmed that recombinant and endogenous eIF3d co-precipitate with endogenous SIN1 but not the mTORC2 components RICTOR and mTOR or the mTORC1 component RAPTOR. It is also shown that the interaction of SIN1 and eIF3d is likely to be occurring in the nucleus. In Hela cells, knockdown of eIF3d expression causes a decrease in the phosphorylation of the mTORC2 targets AKT and PKC on their turn motif sites, but also leads to a significant increase in the phosphorylation and activation of the mTORC1 target S6 kinase. This suggests that eIF3d is a regulator of both mTORC1 and mTORC2 signalling. Reduced expression of eIF3d leads to an increase in cell size, decreased cell proliferation and impairment of cell cycle progression, possibly by promotion of mitotic spindle multipolarity. Additionally, eIF3d knockdown leads to a delay in stress granule formation in Hela cells under arsenite stress, indicating that eIF3d might be acting as a negative mTORC1 regulator by contributing to its stress granule mediated inhibition. Thus eIF3d appears to be a regulator of cell size, proliferation and stress responses, in part mediated by modulation of mTOR activity.
Keyword(s)
Cell Signaling; S6K; eIF3; eIF3d; mTOR