Regulation of Sperm Function by Protein Phosphorylation

描述(申请人提供)：我的实验室的一个长期目标是了解精子运动和受精的生化机制。在精子中，蛋白质合成很少，蛋白质磷酸化是调节细胞功能的主要机制。虽然已知蛋白质磷酸化的变化控制着运动的启动和受精，但我们对调控这些变化的信号通路的了解有限。 这一建议的核心是精子中的一种信号酶，即丝氨酸/苏氨酸磷酸酶PP1Gamma2。它是精子发生和成熟精子功能调节的关键酶。了解这种酶在生化和分子水平上的工作原理将有助于我们理解雄配子的功能。蛋白质纯化和测序使我们能够鉴定出一些潜在的PP1Gamma2结合和调节蛋白。免疫沉淀和与重组蛋白结合的研究表明，其中两个调控蛋白是sds22和蛋白14-3-3-首次在精子中发现。这些在体细胞中具有关键功能的多肽，在调节雄配子功能方面具有新的作用。除了与调节蛋白结合的变化外，PP1Gamma2的磷酸化在附睾精子成熟过程中也会增加。 这一建议的中心假设是，PP1Gamma2的调节作用对精子成熟、运动启动和其他基本精子功能至关重要。我们将使用生化和分子方法在机制水平上表征PP1Gamma2及其调节蛋白之间的磷酸化和结合作用如何与精子运动和受精能力相关。除了对男性配子生物学的基本重要性外，这项研究还在男性不育的诊断和治疗以及开发新的男性避孕方法方面具有潜在的应用价值。

DESCRIPTION (provided by applicant): A long-term goal of my laboratory is to understand the biochemical mechanisms underlying sperm motility and fertilization. In spermatozoa, where little protein synthesis occurs, protein phosphorylation is a major mechanism for regulating cellular functions. While it is known that changes in protein phosphorylation control motility initiation and fertilization, our knowledge of signaling pathways regulating these changes is limited. This proposal centers on a signaling enzyme in spermatozoa, the serine/threonine phosphatase PP1gamma2. It is a key enzyme in spermatogenesis and regulation of mature sperm function. Knowing how this enzyme works at the biochemical and molecular levels will contribute to our understanding of male gamete function. Protein purification and sequencing have enabled us to identify a number of potential PP1gamma2 binding and regulatory proteins. Immunoprecipitation and binding studies with recombinant proteins showed that two of these regulatory proteins are sds22 and protein 14-3-3 - discovered for the first time in spermatozoa. These polypeptides, which have critical functions in somatic cells, have novel roles in regulating male gamete function. In addition to changes in its binding to regulatory proteins, PP1gamma2 phosphorylation is increased during sperm maturation in the epididymis. The central hypothesis in this proposal is that the regulated action of PP1gamma2 is critical for sperm maturation, motility initiation, and other essential sperm functions. We will use biochemical and molecular approaches to characterize, at the mechanistic level, how phosphorylation and binding interactions between PP1gamma2 and its regulatory proteins relate to sperm motility and fertilizing ability. Aside from its basic importance to male gamete biology, this study has potential applications in the diagnosis and treatment of male infertility and in the development of novel approaches to male contraception.