Potential upstream regulators and downstream targets of AMP-activated kinase signaling during oocyte maturation in a marine worm.
Potential upstream regulators and downstream targets of AMP-activated kinase signaling during oocyte maturation in a marine worm.
复制标题
DOI：
10.1530/rep-10-0509
复制
发表时间：
2011-07
期刊：
影响因子：
3.8
通讯作者：
S. A. Stricker
中科院分区：
文献类型：
--
作者：
S. A. Stricker
在小鼠中，卵母细胞成熟的开始（胚泡破裂，GVBD）被cAMP阻断并由AMP激活的激酶（AMPK）触发，与此不同，海洋纽虫Cerebratulus的卵母细胞响应于cAMP升高和AMPK失活而经历GVBD。由于AMPK对哺乳动物或纽虫GVBD的作用的潜在途径尚未完全确定，因此用药理学调节剂处理无卵泡纽虫卵母细胞，随后通过免疫印迹方法使用AMPK的潜在调节剂和靶标的磷酸化特异性抗体进行分析。基于这种磷酸化模式，未成熟的卵母细胞具有活性LKB 1样激酶，磷酸化AMPK的T172位点激活AMPK，而在卵母细胞成熟过程中，AMPK和LKB 1样活性下降。此外，考虑到MAPK可以使体细胞中的AMPK失活，用ERK 1/2 MAPK活化的抑制剂处理卵母细胞。然而，这些试验表明，在成熟相关的AMPK失活过程中T172去磷酸化不需要MAPK，并且观察到的MAPK激酶阻断剂U 0126引起的GVBD抑制实际上是由于异位AMPK激活而不是MAPK失活。类似地，基于使用促成熟因子（MPF）抑制剂的测试，T172去磷酸化发生在MPF激活的上游且独立于MPF激活。另外，MPF和MAPK活性是AMPK上一个可能抑制的S485/491位点完全磷酸化所必需的。此外，在评估可能将AMPK失活与MPF激活联系起来的信号时，获得了成熟卵母细胞上调雷帕霉素靶活性和下调细胞周期蛋白依赖性激酶抑制剂Kip 1的证据。总的来说，这些研究结果进行了讨论相对于多种途径可能介导的AMPK信号在GVBD。
Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMP-activated kinase (AMPK), oocytes of the marine nemertean worm Cerebratulus undergo GVBD in response to cAMP elevations and AMPK deactivation. Since the pathways underlying AMPK's effects on mammalian or nemertean GVBD have not been fully defined, follicle-free nemertean oocytes were treated with pharmacological modulators and subsequently analyzed via immunoblotting methods using phospho-specific antibodies to potential regulators and targets of AMPK. Based on such phosphorylation patterns, immature oocytes possessed an active LKB1-like kinase that phosphorylated AMPK's T172 site to activate AMPK, whereas during oocyte maturation, AMPK and LKB1-like activities declined. In addition, given that MAPK can deactivate AMPK in somatic cells, oocytes were treated with inhibitors of ERK1/2 MAPK activation. However, these assays indicated that T172 dephosphorylation during maturation-associated AMPK deactivation did not require MAPK and that an observed inhibition of GVBD elicited by the MAPK kinase blocker U0126 was actually due to ectopic AMPK activation rather than MAPK inactivation. Similarly, based on tests using an inhibitor of maturation-promoting factor (MPF), T172 dephosphorylation occurred upstream to, and independently of, MPF activation. Alternatively, active MPF and MAPK were necessary for fully phosphorylating a presumably inhibitory S485/491 site on AMPK. Furthermore, in assessing signals possibly linking AMPK deactivation to MPF activation, evidence was obtained for maturing oocytes upregulating target-of-rapamycin activity and downregulating the cyclin-dependent kinase inhibitor Kip1. Collectively, these findings are discussed relative to multiple pathways potentially mediating AMPK signaling during GVBD.