title: L-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways.
abstract: Although amino acids can function as signaling molecules in the regulation of many cellular processes, mechanisms surrounding L-threonine involvement in embryonic stem cell (ESC) functions have not been explored. Thus, we investigated the effect of L-threonine on regulation of mouse (m)ESC self-renewal and related signaling pathways. In L-threonine-depleted mESC culture media mRNA of self-renewal marker genes, [(3)H]thymidine incorporation, expression of c-Myc, Oct4, and cyclins protein was attenuated. In addition, resupplying L-threonine (500 μM) after depletion restores/maintains the mESC proliferation. Disruption of the lipid raft/caveolae microdomain through treatment with methyl-β-cyclodextrin or transfection with caveolin-1 specific small interfering RNA blocked L-threonine-induced proliferation of mESCs. Addition of L-threonine induced phosphorylation of Akt, ERK, p38, JNK/SAPK, and mTOR in a time-dependent manner. This activity was blocked by LY 294002 (PI3K inhibitor), wortmannin (PI3K inhibitor), or an Akt inhibitor. L-threonine-induced activation of mTOR, p70S6K, and 4E-BP1 as well as cyclins and Oct4 were blocked by PD 98059 (ERK inhibitor), SB 203580 (p38 inhibitor) or SP 600125 (JNK inhibitor). Furthermore, L-threonine induced phosphorylation of raptor and rictor binding to mTOR was completely inhibited by 24 h treatment with rapamycin (mTOR inhibitor); however, a 10 min treatment with rapamycin only partially inhibited rictor phosphorylation. L-threonine induced translocation of rictor from the membrane to the cytosol/nuclear, which blocked by pretreatment with rapamycin. In addition, rapamycin blocked L-threonine-induced increases in mRNA expressions of trophoectoderm and mesoderm marker genes and mESC proliferation. In conclusion, L-threonine stimulated ESC G(1)/S transition through lipid raft/caveolae-dependent PI3K/Akt, MAPKs, mTOR, p70S6K, and 4E-BP1 signaling pathways.
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Biotherapy Human Resources Center (BK21), Chonnam National University, Gwangju 500-757, Korea.
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Ryu
authors.forename: Ho Jae
authors.initials: HJ
authors.lastname: Han
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells; D039642:Eukaryotic Initiation Factors; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D051242:Caveolin 1; D018797:Cell Cycle Proteins; D016213:Cyclins; D039642:Eukaryotic Initiation Factors; D020929:Mitogen-Activated Protein Kinase Kinases; D050814:Octamer Transcription Factor-3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D013912:Threonine; D047392:beta-Cyclodextrins; C499527:Eif4ebp1 protein, mouse; C489428:Myc protein, mouse; C494174:Pou5f1 protein, mouse; C108732:methyl-beta-cyclodextrin; C546843:mTOR protein, mouse