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.

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible eIF2α kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a β-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

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