Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation
Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation
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DOI：
10.1128/mcb.17.3.1434
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发表时间：
1997-03-01
影响因子：
5.3
通讯作者：
Bensaude, O
中科院分区：
文献类型：
--
作者：
Bellier, S;Dubois, MF;Bensaude, O
非洲爪蟾卵子发生的特点是一个活跃的转录，成熟后突然停止，调查的转录机制的特点，可能有助于这种转录停滞的变化，RNA聚合酶II最大亚基（RPB 1亚基）的磷酸化状态进行了分析在卵母细胞成熟。我们发现，RPB 1亚基从胚胎发育前期的早期双线期卵母细胞到完全发育的卵母细胞中大量积累，从Dumont IV期到VI期，RPB 1亚基的C-末端结构域（CTD）基本上是低磷酸化的。成熟后，RPB 1亚基的高磷酸化比例急剧增加，在受精后或热休克后1h内，RPB 1亚基的高磷酸化程度明显降低，中期II停滞卵母细胞提取物的CTD激酶活性明显高于前期VI卵母细胞。这种激酶活性的大部分归因于活化的Xp 42促分裂原活化蛋白（MAP）激酶，一种ERK类型的MAP激酶。利用显微注射重组稳定的细胞周期蛋白B1（cyclinB 1）使VI期卵母细胞人工成熟，我们观察到Xp 42 MAP激酶的激活和RPB 1的磷酸化，这两个事件都需要蛋白质合成，这表明p34（cdc 2）的激活激酶不足以离体磷酸化RPB 1，并且与Xp 42 MAP激酶对RPB 1亚基磷酸化的贡献一致。这些结果进一步支持的可能性，即最大的RNA聚合酶II亚基是一个底物的ERK型MAP激酶在卵母细胞成熟过程中，如先前提出的哺乳动物细胞在应激或生长因子刺激。
Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation, To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes, The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophospholylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly, The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes, Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1, Both events required protein synthesis, which demonstrated that activation of p34(cdc2) kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.