Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1.
Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GbetaL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H(2)O(2) on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor-mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H(2)O(2), on the other hand, opposed the effects of insulin by increasing raptor-mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H(2)O(2) on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.
Zhang
Lianqin
L
Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Kimball
Scot R
SR
Jefferson
Leonard S
LS
Shenberger
Jeffrey S
JS
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins; D034741:RNA, Small Interfering / Q000235:genetics; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism*
D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; D006861:Hydrogen Peroxide; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases