Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation.
Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation.
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发表时间：
1996-06
期刊：
影响因子：
4.6
通讯作者：
P. Kalab;J. Kubiak;M. Verlhac;W. Colledge;B. Maro
中科院分区：
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作者：
P. Kalab;J. Kubiak;M. Verlhac;W. Colledge;B. Maro
丝裂原活化蛋白激酶（MAPK）在许多物种卵母细胞减数分裂成熟过程中被激活，然而，其分子靶点仍然未知。这使我们确定了在成熟小鼠卵母细胞和小鼠胚胎的第一个细胞周期中，Mr 82 X 10（3）- 92 X 10（3）（p90 rsk;体细胞中MAPK的主要底物）的核糖体亚基S6激酶的活化。我们评估的磷酸化状态的p90 rsk通过检查电泳迁移率的变化，免疫印迹和测定的激酶活性的免疫沉淀p90 rsk的S6衍生肽。萌发囊泡期（GV）卵母细胞含有一个Mr为82 x 10（3）和84 x 10（3）的双联体，具有较低的S6肽激酶活性（为中期II卵母细胞中最大水平的12%）。在GV击穿（GVBD）后30分钟首次观察到Mr 86 x 10（3）的条带，并在2至3小时内变得突出。在GVBD后1小时，当p90 rsk特异性S6激酶活性达到M II水平的37%时，MAPK没有磷酸化。GVBD后2 h，MAPK磷酸化，p90 rsk激酶活性达到最高水平的86%。当S6肽激酶活性最大时，成熟M II卵母细胞中出现Mr 88 x 10（3）的p90 rsk条带，该条带出现在MAPK磷酸化接近完成时（GVBD后2.5小时）。在激活的卵中，p90 rsk去磷酸化为Mr 86 X 10（3）在原核形成开始后约1小时开始，并持续非常缓慢，直到有丝分裂开始，此时Mr 82 X 10（3）和84 X 10（3）的双联体重新出现。在第一次有丝分裂和诺考达唑阻滞在M期的1-细胞胚胎中，Mr 86 X 10（3）条带的再现表明，M期活化激酶（如p34 cdc 2）在p90 rsk活化中的作用。在成熟的c-mos -/-卵母细胞中没有完全活性的Mr 88 × 10（3）条带，证明了在减数分裂期间p90 rsk的完全激活需要MAPK。第二极体排出后，6-DMAP对激活卵中激酶活性的抑制提供了MAPK和p90 rsk特异性磷酸酶在原核形成前几乎同时激活的证据。
Mitogen-activated protein kinases (MAPK) become activated during the meiotic maturation of oocytes from many species; however, their molecular targets remain unknown. This led us to characterize the activation of the ribosomal subunit S6 kinase of Mr 82 X 10(3) - 92 X 10(3) (p90rsk; a major substrate of MAPK in somatic cells) in maturing mouse oocytes and during the first cell cycle of the mouse embryo. We assessed the phosphorylation state of p90rsk by examining the electrophoretic mobility shifts on immunoblots and measured the kinase activity of immunoprecipitated p90rsk on a S6-derived peptide. Germinal vesicle stage (GV) oocytes contained a doublet of Mr 82 x 10(3) and 84 x 10(3) with a low S6 peptide kinase activity (12% of the maximum level found in metaphase II oocytes). A band of Mr 86 x 10(3) was first observed 30 minutes after GV breakdown (GVBD) and became prominent within 2 to 3 hours. MAPK was not phosphorylated 1 hour after GVBD, when the p90rsk-specific S6 kinase activity reached 37 % of the M II level. 2 hours after GVBD, MAPK became phosphorylated and p90rsk kinase activity reached 86% of the maximum level. The p90rsk band of Mr 88 x 10(3), present in mature M II oocytes when S6 peptide kinase activity is maximum, appeared when MAPK phosphorylation was nearly complete (2.5 hours after GVBD). In activated eggs, the dephosphorylation of p90rsk to Mr 86 X 10(3) starts about 1 hour after the onset of pronuclei formation and continues very slowly until the beginning of mitosis, when the doublet of Mr 82 X 10(3) and 84 X 10(3) reappears. A role for a M-phase activated kinase (like p34cdc2) in p90rsk activation was suggested by the reappearance of the Mr 86 X 10(3) band during first mitosis and in 1-cell embryos arrested in M phase by nocodazole. The requirement of MAPK for the full activation of p90rsk during meiosis was demonstrated by the absence of the fully active Mr 88 X 10(3) band in maturing c-mos -/- oocytes, where MAPK is not activated. The inhibition of kinase activity in activated eggs by 6-DMAP after second polar body extrusion provided evidence that both MAPK- and p90rsk-specific phosphatases are activated at approximately the same time prior to pronuclei formation.