Laboratory of Reproductive and Developmental Biology, Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
Biochemistry. 2011 Jun 28;50(25):5648-59. doi: 10.1021/bi2002696. Epub 2011 Jun 4.
Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.
丝裂原活化蛋白激酶（MAPK）家族成员在非洲爪蟾卵母细胞成熟中发挥重要作用。神经母细胞瘤缺失样激酶（NLK）是一种非典型的 MAPK，已知在脊椎动物和无脊椎动物的多种发育过程中发挥作用，但它在配子发生和配子成熟中的作用尚不清楚。在这项研究中，我们对非洲爪蟾卵母细胞成熟过程中的 NLK1 进行了生化研究。NLK1 在未成熟卵母细胞中表达，其蛋白水平在成熟过程中保持不变。NLK1 在未成熟卵母细胞中无活性，但在成熟过程中被激活，这取决于 Mos 蛋白的合成，但不依赖于 p42 MAPK 的激活。通过注射 5ng mRNA 过量表达 NLK1 可通过翻译激活其 mRNA 加速孕酮诱导的卵母细胞成熟，从而促进 Cyclin B1 蛋白的合成，与卵母细胞中储存的 mRNA 的翻译调控的关键调节因子 Pumilio1（Pum1）、Pumilio2（Pum2）和细胞质多聚腺苷酸化元件结合蛋白（CPEB）的早熟磷酸化一致。通过注射 50ng mRNA 提高 NLK1 的表达水平会诱导 Pum1/Pum2/CPEB 磷酸化、CPEB 降解、Cyclin B1 蛋白合成和无孕酮诱导的卵母细胞成熟。NLK1 在体外磷酸化 Pum1、Pum2 和 CPEB。这些发现为 NLK1 参与非洲爪蟾卵母细胞成熟提供了首个证据。我们认为 NLK1 作为 Mos 的下游激酶发挥作用，催化 Pum1、Pum2 和 CPEB 的磷酸化，以调节包括 Cyclin B1 mRNA 在内的卵母细胞中储存的 mRNA 的翻译。
