Characterization of a Testis Kinase Family (Tssk)

描述（由申请人提供）：蛋白质通过磷酸化的翻译后修饰在许多细胞过程中发挥作用，如细胞外信号的转导，细胞内运输和细胞周期进程。我们的团队对参与精子分化和功能的信号事件的研究感兴趣。虽然减数分裂事件在女性生殖细胞中有很好的特征，但男性生殖细胞减数分裂和分化为成熟精子的调控仍不清楚。最近，在男性生殖细胞中发现了一个新的丝氨酸/苏氨酸激酶家族（睾丸特异性丝氨酸激酶（Tssk））。我们已经证明在成熟哺乳动物精子中存在1个或更多的Tssk家族成员（Hao et al., 2004）（和PR），此外，我们还获得了Tssk同源物在无脊椎动物睾丸中表达的证据。Tssk基因在睾丸中的保守表达模式，以及磷酸化在信号传导过程中的重要性，强烈表明Tssk在生殖细胞分化和/或精子功能中具有重要作用。这一建议旨在为理解Tssk(s)在生殖细胞和成熟精子中的功能奠定基础。该项目的未来方向包括分析Tssk(s)的零突变体（KO），以及利用无脊椎动物模型中可用的遗传方法研究涉及Tssk(s)的信号通路。为了实现这些目标，首先有必要为这些研究创造工具并表征这些激酶。考虑到这一点，本应用将侧重于：1)产生针对Tssk激酶家族5个成员的特异性抗体；2)成熟精子中存在的tsk的鉴定及其亚细胞定位的研究；3) Tssk4激酶活性的生化表征及潜在相互作用伙伴的研究。虽然不是这个应用程序的一部分，但在与NTH的Mitch Eddy博士的合作下，Tssk4的小鼠KO正在生产过程中。重要的是，提出的研究将有助于开始阐明Tssk基因功能的途径。Tssk6 KOs（也称为Sstk）的表型分析最近证实了tsks在精子发生和/或精子功能中的重要性（Spiridonov等人，2005）；这些零突变小鼠在雄性中呈现不育表型，而在雌性中则没有。作为该应用程序的一部分生成的工具对于分析Tssk KO表型和开始Tssk功能的研究非常重要。在精子发生和/或精子功能中预测的任务的重要性表明，这项研究最终可以帮助设计新的人类避孕药。

DESCRIPTION (provided by applicant): Post translational modifications of proteins through phosphorylation play a role in many cellular processes such as the transduction of extracellular signals, intracellular transport, and cell cycle progression. Our group is interested in the study of signaling events that are involved in sperm differentiation and function. Although meiotic events are well characterized in the female germ cells, the regulation of male germ cell meiosis and differentiation into mature sperm is still not well understood. Recently, a novel family of serine/threonine kinases (testes specific serine kinases (Tssk)) has been found post meiotically expressed in the male germ cells. We have demonstrated the presence of 1or more members of the Tssk family in mature mammalian sperm (Hao et al., 2004) (and PR) and in addition, we have obtained evidence that Tssk homologues are expressed in the testes of invertebrates. The conserved testicular expression pattern of Tssk genes, as well as the importance of phosphorylation in signaling processes, strongly suggests that Tssk(s) have important role(s) in germ cell differentiation and/or sperm function. This proposal is designed to set the grounds to understand the function(s) of Tssk(s) in germ cells and in mature sperm. Future directions of this project include the analysis of null mutants (KO) of Tssk(s), as well as the investigation of signaling pathways involving Tssk(s) using genetic methods available in invertebrate models. Toward these goals, it will be necessary first to create tools for these studies and to characterize these kinases. Taking this into consideration, this application will focus on: 1) The generation of specific antibodies against the 5 members of the Tssk kinase family; 2) The identification of the Tssk(s) present in mature sperm and the investigation of their subcellular localization; 3) The biochemical characterization of Tssk4 kinase activity and the study of potential interacting partners. Although not part of this application, in collaboration with Dr. Mitch Eddy from NTH, a mouse KO of Tssk4 is in the process of being produced. Importantly, the proposed research will be useful to begin to illuminate the pathways of Tssk gene function. The predicted importance of Tssks in spermiogenesis and/or sperm function has been recently confirmed by phenotypic analysis of Tssk6 KOs (also known as Sstk)(Spiridonov et al., 2005); these null mutant mice present a sterile phenotype in the males but not in the females. The tools generated as part of this application will be very important for the analysis of Tssk KO phenotypes and to start the study of Tssk function. The predicted importance of Tssks in spermiogenesis and/or sperm function suggests that this research can ultimately help to design novel human contraceptives.