Inactivation of mTor arrests bovine oocytes in the metaphase-I stage, despite reversible inhibition of 4E-BP1 phosphorylation.
The mammalian target of rapamycin (mTor), a Ser/Thr protein kinase, is implicated in the phosphorylation-triggered inactivation of translation repressors, the so-called eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Previous observations in porcine and bovine oocytes revealed an increasing phosphorylation of 4E-BP1 during meiotic maturation. This factor is hypophosphorylated in the germinal-vesicle (GV) stage and its phosphorylation peaks in the metaphase II (M II) stage. In the present approach we intended to block 4E-BP1 phosphorylation specifically to impair initiation of translation and elucidate effects on resumption of meiosis. Torin2, which acts as an active-site mTor inhibitor, reduces 4E-BP1 phosphorylation without any effect on eIF4E and arrests up to 60% of the oocytes in the M I stage. Effects of Torin2 treatment, analyzed by site-specific substrate phosphorylation, were also observed at protein kinase B (Akt or PKB), and cyclin dependent kinases (CDKs). Only minor side effects were found at protein kinase A, C (PKA, PKC), ATM/ATR (Ataxia telangiectasia mutated/AT and Rad3-related protein), and the mitogen activated protein kinases (MAPK) ERK1,2. The inhibition of 4E-BP1 phosphorylation by Torin2 is reversible when cultivating oocytes for additional 24 hr in Torin2-free medium. Even so, oocytes persist in the M I stage. This may indicate the necessity of spatiotemporally regulated translation during meiosis, which cannot be restored later. In conclusion, Torin2 enables an effective and specific inhibition of 4E-BP1 phosphorylation, which may be valuable to investigate maturation specific protein synthesis in more detail.
BVN Neustadt/Aisch, Germany.
Sophia
S
Mayer
Christine
C
Wrenzycki
Wolfgang
W
Tomek
D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D020134:Catalytic Domain / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D005260:Female; D059471:In Vitro Oocyte Maturation Techniques; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D008540:Meiosis / Q000187:drug effects*; D008677:Metaphase / Q000187:drug effects*; D046912:Multiprotein Complexes / Q000187:drug effects; D009287:Naphthyridines / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009866:Oogenesis / Q000187:drug effects*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000032:analysis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000187:drug effects
C558529:9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo(h)(1,6)naphthyridin-2(1H)-one; D002352:Carrier Proteins; D046912:Multiprotein Complexes; D009287:Naphthyridines; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases