Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.
Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.
Tomek
W
W
Research Institute for the Biology of Farm Animals, 18146 Dummerstorf-Rostock, Germany. tomek@fbn-dummerstorf.de
Melo Sterza
F A
FA
Kubelka
M
M
Wollenhaupt
K
K
Torner
H
H
Anger
M
M
Kanitz
W
W
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D007525:Isoelectric Focusing; D016200:Maturation-Promoting Factor / Q000378:metabolism; D008540:Meiosis / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D004676:Myelin Basic Protein / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology*; D010448:Peptide Initiation Factors / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation
D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D005419:Flavonoids; D004676:Myelin Basic Protein; D010448:Peptide Initiation Factors; D019319:Okadaic Acid; D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases; D010744:Phosphoric Monoester Hydrolases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one