Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.
Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.
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DOI：
10.1371/journal.pone.0141961
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Vijayaraghavan S
中科院分区：
文献类型：
--
作者：
Dudiki T;Kadunganattil S;Ferrara JK;Kline DW;Vijayaraghavan S
哺乳动物精子含有丝氨酸/苏氨酸磷酸酶Pp1、γ2和PP2A。精子蛋白Pp1、γ-2的作用已得到较好的研究。在这里，我们证实了PP2A在精子中的存在，并表明在附睾精子成熟过程中，它经历了甲基化(亮氨酸309)、酪氨酸磷酸化(酪氨酸307)和催化活性的显著变化。从附睾头近端、附睾头远端和附睾尾分离的精子含有等量的PP2A免疫反应。利用去甲基化敏感抗体，我们发现PP2A在附睾头末端和尾部的精子中发生甲基化，而在附睾头近端的精子中不发生甲基化。用微囊藻毒素琼脂糖凝胶分离PP2A，然后进行碱处理，使蛋白质的羧甲酯发生水解，从而证实了PP2A的甲基化状态。精子PP2A的酪氨酸磷酸化与甲基化成反比。也就是说，PP2A在去甲基化时被酪氨酸磷酸化，但在甲基化时没有。用L同型半胱氨酸和腺苷处理精子也会影响PP2A去甲基化及其相互作用的酪氨酸磷酸化，已知这两种药物会升高细胞内S-腺苷同型半胱氨酸，这是一种甲基转移酶反馈抑制剂。PP2A的催化活性在附睾精子成熟过程中下降。冈田酸或L-同型半胱氨酸和腺苷共同孵育精子后，精子运动参数如精子运动百分率、精子运动速度和精子头部侧向振幅均显著增加。PP2A去甲基化或药物抑制也会导致糖原合成酶激酶-3(GSK3)的磷酸化增加。我们的结果首次表明，由于甲基化和酪氨酸磷酸化导致的PP2A活性在精子中发生了变化，这些变化可能在精子功能的调节中发挥重要作用。
Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.