Regulation Of MTOR Signaling By Reactive Oxygen Species And Its Mechanisms

The mammalian target of rapamycin（mTOR） is a serine/threonine kinase whose activity can be inhibited by rapamicin binding with FKBP12（FK506 binding protein 12）.mTOR exists in two heteromeric complexes,mTORC1and mTORC2,mTORC1 consists of the three proteins mTOR,mLST8（also known asGβL） and Raptor（for regulatory associated protein of mTOR）,and largely controls translation,transcription, cell growth,proliferation,autophagy,ribosome and biogenisis in response to growth factors,insulin,nutrients,energy availability and cellular stressors such as hypoxia, osmotic stress,reactive oxygen species and viral infection,mTORC2 consists of mTOR,mLST8,Rictor（for rapamycin-insensitive companion of mTOR） and Sin1（SAPK interacting protein 1）,and has been shown to control actin cytoskeleton dynamics and Akt（473） phosphorylation.The regulatory mechanism of mTORC2 remains unclear.An increasing number of human diseases have been linked to the dysregulation of mTOR,including immunological disorders,cancer,metabolic diseases,cardiovascular diseases and neurological disorders.Insulin or growth factors initiate signaling at their receptors,leading to activation of PI3K（phosphatidylinositol 3-kinase）.The lipid products of PI3K,PIP3 （phosphatidylinositol 3,4,5 trisphosphate）,recruit both PDK1（PI3K-dependent kinase 1,PKD1） and Akt to the plasma membrane.Akt is subsequently phosphorylated on T308 by PDK1 and S473 by mTORC2,leading to full activation. Akt then phosphorylates numerous targets to promote growth and survival,including the TSC1/2 complex.By phosphorylating TSC2,Akt inactivates TSC2 GAP activity for the small G protein Rheb.GTP-bound Rheb is a potent activator of the rapamycin-sensitive complex mTORC1,mTORC1 phosphorylates several targets, including the translation control proteins ribosomal protein S6 kinase-1（S6K1） and elongation factor 4E binding protein 1（4E-BP1）,which are components of the translational control machinery and mediate ribosome biogenesis and cap-dependent translation,respectively.The phosphorylation of S6K1 on T389 and 4EBP1 on T37/T46 are often used as functional readouts of mTOR activity,since these sites are specifically phosphorylated by mTOR both in vitro and in vivo and are inhibited by rapamycin treatment.Reactive oxygen species（ROS） are involved in many signal pathways and either induces cell proliferation or apoptosis.Oxidative stress is linked to many human diseases such as cancer,cardiac failure,cardiac hypertrophia,artherosclerosis, diabetes,hypertension and osteoporosis.Reactive oxygen species have been found to either activate or inhibit mTORC 1 by unknown mechanisms.There are no reports on the regulation of mTORC2 by ROS till now.In this essay,we explored the mechanisms through which ROS regulating mTORC1 and mTORC2 by using alloxan-treated mouse,and ulatra violet and hydrogen peroxide-treated variety of cells as models.We found that:Firstly,reactive oxygen species could either activate or inhibit mTORC1 signaling contingent on the cell type,exposure length,dose/concentration of the stressor and time between stress stimulus.In one hand,reactive oxygen species could activate mTORC1 signaling.UV light exposure stimulated S6K1（T389） activity by inducing the generation of ROS. The concentration of hydrogen peroxide and MDA increased distinctly in the homogenates of liver,kidney,brain,heart and skeletal muscle 72 h after 150mg/kg alloxan intraperitoneal ingection to mice,acompanied with increasing mTORC1 activity（S6K1（T389）,S6（S235/236） ）.In some cells,such as mouse osteoblast cell line MC3T3-E1,mouse embryonic fibroblast（MEF） and human breast cancer cell line MCF-7,low doses of hydrogen peroxide（H₂O₂） activated mTORC1 dose-and time- dependently.But the minimal doses and interval of hydrogen peroxide were different from various cells.In other cells,such as human embryonic kidney cell line HEK293,human osteosarcoma MG63,primary mouse bone marrow stromal cells （BMSCs） and osteoblast,however,we could not detect any increase of mTORC1 activity upon different concentrations of H₂O₂ stimulation..In the other hand,reactive oxygen species suppressed mTORC1 signaling in all cells that we screened.High doses of hydrogen peroxide suppressed mTORC1 signaling dose-and time- dependently while the minimal dose and interval of hydrogen peroxide were different from various cells.Secondly,how did ROS activate or suppress mTORC1 signaling?The activation of mTORC1 by low doses of H₂O₂ was blocked by LY294002,an inhibitor of PI3K,suggesting that PI3K was responsible for the process.S6K1 was activated both in TSC2 null MEF cell line(TSC2^-/-/P53^-/-) and in wild type MEF (TSC2^+/+P53^-/-),suggesting that TSC2 was not involved in this process.The suppression of mTORC1 by high doses of H₂O₂ could be reversed by OKA, an inhibitor of protein phosphatase 2A（PP2A）,suggesting that PP2A is responsible for the process.Furthermore,H₂O₂ stimulates association of endogenous S6K1 with catalytic subunit of PP2A（PP2Ac）.Low doses of H₂O₂ prevent cell proliferation and induce G2 cell cycle arrest,while high doses of H₂O₂ induce cell death.OKA could reverse H₂O₂-induced G2 arrest and significantly decrease H₂O₂-induced cell death. Taken together,it is suggested that PP2A mediates mTORC 1 inhibition by H₂O₂ and that PP2A is involved in cell cycle arrest and cell death induced d by H₂O₂.H₂O₂ stimulates phosphorylation of AMPK（T172） and Raptor（S792） dose- and time-dependently.Compound c,a AMPK-specific inhibitor,inhibits H₂O₂-stimulated AMPK（T172） and Raptor（S792） phosphorylation,and reverses H₂O₂-induced de-phosphorylation of S6K1（T389）,S6（S235/236） and 4E-BP1（T37/46）.It is suggested that AMPK and Raptor is responsible for the suppression of mTORC1 signaling by H202. mTORC 1 activity was inhibited by high concentrations of H₂O₂ either in TSC2^-/- P53^-/- MEF or in TSC2^+/+ P53^-/- MEF.Furthermore,H₂O₂ did not enhance the association of TSC2 with AMPK.It demonstrates that TSC2 is not related to this process.Rheb is an upstream activator of mTOR.Overexpression of wild type or active mutant of Rheb（Q64L） prevents amino acid starvation-induced decrease of mTORC1 activity,but does not prevent H₂O₂ inhibition of mTORC1.It is suggested that Rheb is not involved in the regulation of mTOR by H₂O₂.Lastly,NFκB/IKK and PI3K may be involved in the regulation of mTORC2 by H₂O₂.mTORC2 function as kinase of PKB/Akt,which contributes to phosphorylation of Akt on Ser 473.Akt（S473） was found to be activated by H₂O₂ rapidly and dramatically.As the upstream of mTORC2 was unknown,inhibitors of ERK、NFκB/IKK、PI3K、PTK、PP2A,which were found relating to reactive oxygen species,including,were used in our study.We found that inhibitors of NFκB/IKK and PI3K could block phosphorlation of AKT（S473） by H₂O₂,indicating that the two passways may mediate this process.In a summary,reactive oxygen species could either activate or inhibit mTORC1 signaling in a time-,dose- and cell type- dependent manner.Activation of mTORC1 signaling was dependent on PI3K,but not TSC2.Suppression of mTORC1 was related to PP2A,phosphorylation of Raptor by AMPK,but not TSC2 and Rheb. Hydrogen peroxide activated mTORC2（Akt（S473）） by IKK.These findings provide new data for mTOR signaling network and targets for diseases which ROS and mTOR are involved in.