Abstract
Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.
Highlights
Data indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling
Extensive data suggest that mTORC1 mediates the phosphorylation of Thr-37/46 in 4E-BP, because this is impaired by inhibitors of the kinase activity of mTOR, by the tuberous sclerosis complex (TSC1/2), a negative regulator of Rheb and mTORC1, and by decreasing the cellular levels of mTOR or the mTORC1 component raptor [6, 7]
That report suggested that Rheb1⁄7GTP bound to FKBP38, releasing the latter protein from mTOR and thereby eliciting the Chinese hamster ovary (CHO) cells were grown as described activation of mTORC1
Summary
FKBP38 ( termed FKBP8 [16]) is an immunophilin and belongs to the peptidyl/prolyl cis-trans-isomerase protein family. FKBP38 was reported to bind to Rheb, such that Rheb1⁄7GTP induced the release of FKBP38 from mTOR This would provide a mechanism by which Rheb1⁄7GTP could activate mTORC1 signaling (Fig. 1B). It is not clear from the available data whether FKBP38 is involved in the regulation of mTORC1 by amino acids [24]. The fact that starving cells for amino acids (especially leucine) leads to inhibition of mTORC1 signaling has led to the suggestion that uncharged tRNA, generated because of insufficiency of amino acids, might act as a proximal negative regulator of mTORC1 [29]. It is formally possible that activated mGcn can negatively regulate mTORC1 signaling, perhaps by phosphorylating mTORC1 or a regulator of this complex Given the high level of interest in mTORC1 signaling, it was important to assess the roles of these proposed regulators of mammalian TORC1 signaling, including their possible importance for the control of this protein kinase by amino acids
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