CDK7 and CCNH Are Components of CDK-Activating Kinase and Are Required for Meiotic Progression of Pig Oocytes
CDK7 and CCNH Are Components of CDK-Activating Kinase and Are Required for Meiotic Progression of Pig Oocytes
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DOI：
10.1095/biolreprod.111.091801
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发表时间：
2011-12-01
影响因子：
3.6
通讯作者：
Naito, Kunihiko
中科院分区：
文献类型：
--
作者：
Fujii, Wataru;Nishimura, Takanori;Naito, Kunihiko
CDK激活激酶（CAK）磷酸化CDC 2的苏氨酸161（T161），其是成熟/M期促进因子（MPF）的催化亚基，并且是有丝分裂中MPF激活所必需的。CAK被认为由催化亚基、调节亚基和组装因子组成：分别为CDK 7、CCNH（也称为细胞周期蛋白H）和MNAT 1（也称为MAT 1）。尽管已经知道卵母细胞减数分裂进程受MPF活性的调节，但CAK在减数分裂中的作用仍不清楚。在本研究中，我们试图证实CAK参与猪未成熟卵母细胞减数分裂进程。CDC 2的T161磷酸化在胚泡破裂（GVBD）前后及其后培养18 ~ 48 h出现。在18小时的GVBD率增加的过表达的CDC 2，但不是突变的CDC 2（T161被丙氨酸取代）。CDK 7，CCNH和MNAT 1的转录本在整个培养期间都是可检测的，并且它们在卵母细胞成熟过程中的蛋白质分布模式与有丝分裂体细胞中报道的相同。CDK 7或CCNH的过表达加速了减数分裂事件，例如减数分裂恢复、CDC 2的T161磷酸化、CCNB（也称为Cyclin B）合成和MPF活化。相反，CDK 7或CCNH的敲低导致这些减数分裂事件的抑制。相比之下，MNAT 1的过表达和反义RNA注射对减数分裂恢复、CDC 2的T161磷酸化状态或MPF活性没有影响。这些结果表明，CDK 7和CCNH通过T161磷酸化激活CDC 2，并组成CAK，这是猪卵母细胞成熟过程中正常减数分裂进程所必需的。
CDK-activating kinase (CAK) phosphorylates threonine 161 (T161) of CDC2, a catalytic subunit of maturation/M-phase promoting factor (MPF), and is essential for MPF activation in mitosis. CAK has been thought to consist of a catalytic subunit, a regulatory subunit and an assembly factor: CDK7, CCNH (also known as cyclin H), and MNAT1 (also known as MAT1), respectively. Although it is known that the meiotic progression of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in the meiotic progression of porcine immature oocytes. The T161 phosphorylation of CDC2 was found around germinal vesicle breakdown (GVBD) and thereafter from 18 to 48 h of culture. The GVBD rate at 18 h was increased by the overexpression of CDC2 but not mutated CDC2 (T161 replaced by alanine). Transcripts of CDK7, CCNH, and MNAT1 were detectable throughout the culture period, and their protein distribution patterns during oocyte maturation were the same as those reported in mitotic somatic cells. Overexpression of CDK7 or CCNH accelerated the meiotic events, such as meiotic resumption, T161 phosphorylation of CDC2, CCNB (also known as Cyclin B) synthesis, and MPF activation. On the contrary, knockdown of CDK7 or CCNH caused the inhibition of these meiotic events. In contrast, overexpression and antisense RNA injection of MNAT1 had no influence on meiotic resumption, the status of T161 phosphorylation of CDC2, or MPF activity. These results suggest that CDK7 and CCNH activate CDC2 by T161 phosphorylation and make up CAK, which is required for normal meiotic progression during porcine oocyte maturation.