Phosphorylation Events During Sperm Capacitation

描述（由申请人提供）：哺乳动物精子在射精后不能立即使卵子受精。它们在雌性生殖道中停留一段时间后获得受精能力。雌性生殖道发生的生理变化使精子能够受精，构成“精子获能”现象（Austin，1952; Chang，1951）。使用小鼠作为实验模型，我们已经证明获能与蛋白质子集的酪氨酸（tyr）磷酸化的增加相关（Visconti等人，1995年a）。我们还证明了蛋白质tyr磷酸化的增加以及获能受cAMP依赖性途径调节（Visconti等人，1995年b）。该调节途径的存在随后在来自包括人的其它物种的精子中得到证实（Leclerc等人，1996; Osheroff等人，1999年）。尽管在理解获能过程中调节磷酸化的机制方面取得了这些进展，但对这种磷酸化级联反应的蛋白质靶点以及参与精子功能调节的激酶和磷酸酶的身份知之甚少。最近，我们使用了与串联质谱（MS/MS）分析相结合的二维（2D）聚丙烯酰胺凝胶电泳（PAGE）方法（Ficarro等人，2003）以鉴定在人类精子获能期间经历Tyr磷酸化的蛋白质。在相同的工作中，我们已经通过MS/MS分析了来自获能人精子群体的蛋白质中的确切磷酸化序列，并鉴定了tyr以及丝氨酸（ser）和苏氨酸（thr）中的磷酸化序列（Ficarro等人，2003年）。其中的蛋白质目标，valosin-containing蛋白（VCP），从同一个家庭的SNARE相互作用蛋白N-乙基马来酰亚胺可溶性因子（NSF）的ATP酶被发现在获能过程中酪氨酸磷酸化。此外，VCP的免疫定位显示伴随精子获能的荧光模式的变化。联合收割机结合了上述发现，并假定在获能过程中特异性磷酸化的蛋白质以及负责其磷酸化的激酶是调节哺乳动物精子获能所必需的。这个提议的目的是进一步阐明哺乳动物精子中控制蛋白质磷酸化级联反应的顺序。磷酸化蛋白质底物和参与精子功能调节的激酶的表征将为受精过程的药理学控制提供新的靶点。

DESCRIPTION (provided by applicant): Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes occurring in the female reproductive tract rendering the sperm able to fertilize constitute the phenomenon of "sperm capacitation" (Austin, 1952; Chang, 1951). Using the mouse as an experimental model, we have demonstrated that capacitation is associated with an increase in the tyrosine (tyr) phosphorylation of a subset of proteins (Visconti et al., 1995a). We have also demonstrated that the increase in protein tyr phosphorylation as well as capacitation are regulated by a cAMP-dependent pathway (Visconti et al., 1995b). The presence of this regulatory pathway has subsequently been demonstrated in sperm from other species including human (Leclerc et al., 1996; Osheroff et al., 1999). Despite these advances in understanding the mechanisms regulating phosphorylation during capacitation, little is known about the identity of the protein targets of this phosphorylation cascade and of the kinases and phosphatases involved in the regulation of sperm function. Recently, we have used a 2 dimensional (2D) polyacrylamide gel electrophoresis (PAGE) approach combined with tandem mass spectrometry (MS/MS) analysis (Ficarro et al., 2003) to identify proteins that undergo tyr phosphorylation during capacitation of human sperm. In the same work, we have analyzed the exact phosphorylated sequence by MS/MS in proteins from a population of capacitated human sperm and identified sequences phosphorylated in tyr as well as in serine (ser) and threonine (thr) (Ficarro et al., 2003). Among the protein targets, valosin-containing protein (VCP), an ATPase from the same family of the SNARE-interacting protein N-ethyl maleimide soluble factor (NSF) was found to be tyr phosphorylated during capacitation. In addition, immunolocalization of VCP showed a change in fluorescent pattern accompanying sperm capacitation. The hypotheses underlying this proposal combine the above set of findings and postulate that proteins specifically phosphorylated during capacitation as well as the kinases responsible for their phosphorylation are required for the regulation of mammalian sperm capacitation. The objective of this proposal is to further elucidate the sequence of reactions that control protein phosphorylation cascades in mammalian sperm. Characterization of the phosphorylated protein substrates and the kinase(s) involved in the regulation of sperm function will provide novel targets for pharmacological control of the fertilization process.