Endosomal docking of mTOR modulates mTORC1 activity

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase, evolutionary conserved from yeast to human, which forms two functional distinct and independently regulated multiprotein complexes, dubbed mTORC1 and mTORC2. mTORC1 contains mTOR, raptor, mLST8, PRAS40 and DEPTOR, and is sensitive to the immunosuppressive and anti-cancer drug rapamycin. mTORC1 regulates translation via phosphorylation of S6K1 and 4E-BP1, two well-characterized effector proteins. mTORC2 consists of mTOR, rictor, mLST8, mSIN1, protor and DEPTOR, and is rapamycin-insensitive. It regulates the organization of the actin cytoskeleton and is involved in cell survival.
Both mTORC1 and mTORC2 are stimulated by growth factors, but only mTORC1 is controlled as well by cellular energy status and nutrients such as amino acids. How cells sense amino acid sufficiency to control protein synthesis and eventually cell growth is largely unknown. Amino acid deprivation results in rapid dephosphorylation of S6K1 and 4E-BP1, while amino acid stimulation leads to the rapid mTORC1-mediated phosphorylation of these molecules. Moreover, amino acid stimulation triggers the re-localization of mTORC1 to late endosomes/lysosomes, where Rheb is supposed to be localized and thereby allowing Rheb to directly interact with and activate mTORC1. Additionally, vacuolar protein sorting 34 (Vps34), the sole class III phosphatidylinositol 3-kinase (PI3K), and its phospholipid product phosphatidylinositol 3-phosphate [PtdIns(3)P] have also been implicated in mTORC1 activation. However, concerns exist in connecting Vps34 with mTORC1 regulation. Therefore, we have decided to investigated in more depth the role of Vps34 and PtdIns(3)P in the amino acid pathway to mTORC1. Ectopic expression of either wild type or kinase-dead human Vps34 (hVps34) did not change mTORC1 activity. Deletion of hVps34 by RNAi did not result in a complete decrease of mTORC1 activation, indicating that another PI3K might generate PtIns(3)P. Depletion of the class II PI3K isoforms PI3K-C2alpha and PI3K-C2beta, which have been demonstrated to generate PtdIns(3)P as well, leads to an isoform specific regulation of mTORC1 activity. PI3K-C2alpha deletion increased, while PI3K-C2beta knockdown only partially reduced mTORC1 activity. However, by directly affecting cellular PtdIns(3)P availability either by sequestering PtdIns(3)P with a tandem FYVE domain (Fab1/YOTB/Vac1/EEA1; named after the first four proteins in which it was identified) or by acute treatment with exogenous PtdIns(3)P, mTORC1 activity almost completely decreased or strongly increased, respectively. These results confirm the requirement of PtdIns(3)P in the mTORC1 pathway.
Consistent with the requirement of PtdIns(3)P in endosomal homeostasis, we reasoned that PtdIns(3,5)P2, which is generated by PIKfyve using PtdIns(3)P as substrate and required for early to late endosome trafficking, might as well be implicated in the amino acid-dependent pathway to mTORC1. Pharmacologic inhibition of PIKfyve strongly decreased mTORC1-mediated phosphorylation of S6K1 without affecting the catalytic activity of mTOR and PtdIns(3)P generation, and abrogated the translocation of mTOR to late endosomes indicating that the inhibiting effect on mTORC1, induced through the inhibition of PIKfyve, is probably due to a defect in endosomal membrane transport to late endosomes.
We are the first to show that PtdIns(3,5)P2, generated by PIKfyve, is involved in mTORC1 activation and moreover, demonstrating that an unperturbed endosomal transport is required for late endosomal re-localization of mTORC1 induced by amino acids.
However, the above described result cannot be generalized for different cell lines. While in HEK293 cells PIKfyve inhibition strongly blocked mTORC1 activity, in A375 cells, an aggressive melanoma cell line, the inhibition was less pronounced. This can be explained that A375 cells have a constitutive active MAPK pathway due to B-Raf V600E mutation, which impinges on mTORC1 activity. Therefore, this indicates that cancer cells with mutated MAPK or PI3K-Akt/PKB pathway cannot be treated solely with the PIKfyve inhibitor but need additional treatments.