Huang Zaohua, Myers Kimberley, Khatra Balwant, Vijayaraghavan Srinivasan
Department of Biological Sciences, Kent State University, Kent, Ohio 44242, USA.
Biol Reprod. 2004 Jul;71(1):177-84. doi: 10.1095/biolreprod.104.027284. Epub 2004 Mar 17.
The protein phosphatase PP1gamma2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1gamma2 is phosphorylated and that the amount of phosphorylated PP1gamma2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1gamma2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species-bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1gamma2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1gamma2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1gamma2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1gamma2 is one of its binding partners.
蛋白磷酸酶PP1γ2在精子活力和生育能力的调节中起关键作用。其活性受其结合蛋白和磷酸化作用的调节。我们最近发现PP1γ2会发生磷酸化，并且在精子附睾成熟过程中磷酸化的PP1γ2含量会增加（Huang等人，《生物繁殖》2004年；70:439 - 447）。微量测序显示，在牛精子提取物的柱层析过程中，蛋白14 - 3 - 3与磷酸化的PP1γ2一同洗脱。蛋白质印迹分析证实，蛋白14 - 3 - 3不仅存在于牛精子中，也存在于多种物种的精子中，包括公牛、仓鼠、鲎、猴子、大鼠、火鸡和非洲爪蟾。通过免疫共沉淀实验以及用重组GST - 14 - 3 - 3进行的下拉实验，证实了PP1γ2与蛋白14 - 3 - 3之间的结合。蛋白质印迹分析以及用针对蛋白14 - 3 - 3共有结合结构域的抗体进行的蛋白14 - 3 - 3免疫沉淀显示，除了PP1γ2之外，精子中至少还存在另外两种蛋白14 - 3 - 3结合伴侣。荧光免疫细胞化学结果表明，磷酸化的PP1γ2和蛋白14 - 3 - 3都定位于牛精子头部顶体后区域和主段。这些结果共同提供了确凿证据，证明蛋白14 - 3 - 3存在于成熟精子中，且PP1γ2是其结合伴侣之一。
