Bub1 is activated by the protein kinase p90Rsk during Xenopus oocyte maturation
Bub1 is activated by the protein kinase p90Rsk during Xenopus oocyte maturation
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DOI：
10.1016/s0960-9822(01)00045-8
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发表时间：
2001-02-06
期刊：
影响因子：
9.2
通讯作者：
Maller, JL
中科院分区：
文献类型：
--
作者：
Schwab, MS;Roberts, BT;Maller, JL
背景：动粒连接(纺锤体组装)检查点阻止中期细胞退出有丝分裂，直到所有染色体在中期平板上正确排列。该检查点通过阻止后期促进复合体(APC)的激活来运作，APC通过降解有丝分裂周期蛋白和其他蛋白质来触发后期。在酵母中，丝氨酸/苏氨酸蛋白激酶Bub1和WD-Repeat蛋白Bub3是调节着丝粒附着检查点的信号转导级联的元件。在哺乳动物细胞中，在有丝分裂过程中，激活的MAPK存在于动粒上，其活性被纺锤体组装检查点上调。在脊椎动物未受精卵中，一种特殊形式的减数分裂中期停滞是由蛋白激酶P90(RSK)的MAPK激活所介导的，从而导致对APC的抑制，然而，目前尚不清楚P90(RSK)引起的依赖于CSF的中期停滞是否涉及纺锤体组装检查点的组成部分。在非洲爪哇卵母细胞中，Bub1在减数分裂I和减数分裂II期间都定位于动粒，而在减数分裂I期间，Bub1的凝胶迁移率降低，反映了该酶的磷酸化和激活。通过选择性地刺激MAPK途径，可以在间期鸡蛋提取液中诱导Bub1的激活。在用MEK1抑制剂U0126处理的卵母细胞中，MAPK通路不会被激活，Bub1仍然处于低活性、未移位的形式。在U0126存在的情况下，注射MAPK的结构性活性靶点蛋白激酶P90(RSK)可以恢复Bub1的激活。此外，纯化的p90(RSK)在体外可磷酸化Bub1，并提高其蛋白激酶活性。结论：在非洲爪哇减数分裂过程中，动粒连接检查点上游成分Bub1以MAPK依赖的方式被激活。此外，MAPK的单一底物P90(RSK)在体内外足以激活Bub1。这些结果表明，在脊椎动物卵中，动粒附着/纺锤体组装检查点蛋白，包括Bub1，位于P90(RSK)下游，可能是P90(RSK)抑制APC和依赖于CSF的中期酶抑制的效应者。
Background: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle, In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC, However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint.Results: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bubl upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bubl can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bubl remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bubl in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bubl in vitro and increases its protein kinase activity.Conclusions: Bubl, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is, sufficient to activate Bubl in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bubl, are downstream of p90(Rsk) and may be effecters of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).