Huang Zaohua, Vijayaraghavan Srinivasan
Biological Sciences Department, Kent State University, Kent, Ohio 44242, USA.
Biol Reprod. 2004 Feb;70(2):439-47. doi: 10.1095/biolreprod.103.020024. Epub 2003 Oct 20.
The enzyme PP1gamma2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1gamma2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1gamma2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1gamma2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1gamma2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1gamma2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1gamma2 is catalytically active. Phosphorylated PP1gamma2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1gamma2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1gamma2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1gamma2 in signaling events during fertilization.
酶PP1γ2是1型蛋白磷酸酶（PP1）的睾丸和精子特异性同工型，也是精子中PP1的唯一同工型。酶PP1γ2对精子发生至关重要，也是精子运动发育和调节中的关键酶。该酶的羧基末端含有细胞周期蛋白依赖性激酶磷酸化的共有氨基酸序列。使用针对该磷酸化氨基酸序列结构域的特异性抗体，我们发现磷酸化的PP1γ2存在于牛附睾精子中。与不动的附睾头精子相比，活动的附睾尾精子中磷酸化PP1γ2的水平显著更高。一些刺激精子运动的处理，如2-氯腺苷、cAMP类似物、cAMP磷酸二酯酶抑制剂和钙，并没有改变磷酸化PP1γ2的水平。然而，作为蛋白磷酸酶亚型PP1和PP2A抑制剂的花萼海绵诱癌素A，显著增加了附睾头和附睾尾精子中磷酸化PP1γ2的水平。通过柱色谱法进行部分纯化表明，磷酸化的PP1γ2具有催化活性。磷酸化的PP1γ2是附睾尾精子提取物中该酶唯一的自发催化活性形式。蛋白质印迹分析表明，细胞周期蛋白依赖性激酶2这种在PP1同工型羧基末端磷酸化共有结构域的酶之一存在于精子中。对从纯化的精子头部和尾部片段中提取的蛋白质进行蛋白质印迹分析表明，磷酸化的PP1γ2主要存在于精子头部。荧光免疫细胞化学也表明，磷酸化的PP1γ2主要存在于精子头部的后部区域。其在精子成熟过程中独特的亚细胞定位及其水平变化表明，精子磷酸化的PP1γ2在受精过程中的信号事件中可能发挥作用。
