Cytoplasmic Retention as a Regulatory Mechanism in Embryogenesis

细胞质保留作为胚胎发生的调节机制

• 批准号: 9603948
• 负责人: Laurence Etkin
• 金额: $ 33万
• 依托单位: University of Texas, M.D. Anderson Cancer Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9603948&HistoricalAwards=false
• 关键词: Cytoplasmic; Retention; Regulatory; Mechanism; Embryogenesis

Etkin 9603948 Cytoplasmic retention is an important regulatory step controlling the function of nuclear proteins. It is a universal phenomenon occurring in a variety of systems from yeast to man and regulating the comDartmentalkation of proteins during the cell cycle, different stages of differentiation, and sDecHic stages of development. During development cytoplasmic retention was observed with the dorsal gene product in Drosophila (Govind and Steward, 1992) and the late shifting maternal proteins in Xenopus (Dreyer et al., 1983; Miller et al., 1989,1991). Dr. Etkin is analyzing one of the late shifting proteins, Xenopus nuclear factor 7 (xnf7), which originates in the oocyte nucleus (GV) and is retained in the cytoplasm until it enters the nucleus at the mid blastula transition (MBT) (Miller et al., 1989,1991; Reddy et al.,1991). Xnf7 is a member of a novel zinc finger gene family consisting of protoonconenes and transcription factors (Ready and Etkin, 1991; Reddy et al., 1992; Freemont, 1993) and was recently shown to function in the dorsaVventral patterning of the embryo (El-Hodiri et al, submitted). Li et al (1994a) demonstrated the presence of a 22 amino acid cytoplasmic retention domain (CRD) that requires the phosphorylation of two phosphorylation sites (sites 1 and 2) to function in the cytoplasmic retention of xnf7 (Shou et al., 1996). Dr. Etkin has also shown that cytoplasmic retention of xnf7 most likely involves an anchor mechanism (Li et al.,1994a) and that the protein exists in a 670kD Drotein complex consisting of xnf7 and two other proteins (Shou et al., 1996; Kuang, Che, Elhodiri and Etkin, unpublished). His hypothesis is that the retention of xnf7 in the cytoplasm prior to the MBT is the result of phosphorylation of site 1 and site 2 by specific kineses that influence the interaction of the CRD with a cytoplasmic anchor complex. In the present proposal he outlined a series of experimental approaches aimed testing this hypothesis and characterizing the compon ents of the anchor complex by addressing the following specific aims: 1.To identify the kineses that phosphorylate sites 1 and 2 which regulate cytoplasmic retention; 2. To identify xnf7 interacting proteins (XIP) using both biochemical and genetic approaches; 3.To perform a functional anaTysis of the XIP proteins. He believes that the analysis of this phenomenon using xnf7 as a model will serve as a paradigm for understanding the basic principles of the cytoplasmic retention mechanism in many systems. In addition, identification of the protein kineses that regulate the CRD function in retention will reveal the nature of the signal transduction pathways regulating this process. This will be important in integrating cytoplasmic retention of xnf7 with other biological processes such as axial patterning and cell cycle regulation which are also controlled through signal transduction pathways.

埃特金9603948 胞质滞留是控制核蛋白功能的重要调节步骤。它是一种普遍现象，存在于从酵母到人类的各种系统中，并在细胞周期、分化的不同阶段和发育的sDecHic阶段调节蛋白质的合成。在发育期间，在果蝇中观察到背侧基因产物的细胞质滞留（Govind和Steward，1992），在非洲爪蟾中观察到晚期转移的母体蛋白（Dreyer等人，1983;米勒等人，1989，1991）。 Etkin博士正在分析一种晚期转移蛋白，非洲爪蟾核因子7（xnf 7），它起源于卵母细胞核（GV），并保留在细胞质中，直到它在囊胚中期过渡（MBT）进入细胞核（米勒等人，1989，1991; Reddy等人，1991年）。Xnf 7是由原癌基因和转录因子组成的新型锌指基因家族的成员（Ready和Etkin，1991; Reddy等人，1992; Freemont，1993），并且最近显示在胚胎的背腹图案中起作用（El-Hodiri等人，提交）。Li等（1994 a）证明存在22个氨基酸的胞质滞留结构域（CRD），其需要两个磷酸化位点（位点1和2）的磷酸化以在xnf 7的胞质滞留中起作用（Shou等，1996年）。Etkin博士还表明，xnf 7的细胞质保留最有可能涉及锚机制（Li et al.，1994 a），并且该蛋白存在于由xnf 7和两种其它蛋白组成的670 kD Drotein复合物中（Shou等人，1996; Kuang，Che，Elhodiri and Etkin，unpublished）.他的假设是，在MBT之前，xnf 7在细胞质中的保留是通过影响CRD与细胞质锚复合物相互作用的特定激酶使位点1和位点2磷酸化的结果。在目前的建议中，他概述了一系列实验方法，旨在测试这一假设，并通过解决以下具体目标来表征锚复合物的组分：1.鉴定磷酸化调节细胞质保留的位点1和2的激酶;利用生物化学和遗传学方法鉴定xnf 7相互作用蛋白（XIP）; 3.对XIP蛋白进行功能分析。他认为，以xnf 7为模型对这一现象的分析，将为理解许多系统中细胞质滞留机制的基本原理提供范例。此外，确定的蛋白质激酶，调节CRD功能的保留将揭示调节这一过程的信号转导途径的性质。这对于整合xnf 7的细胞质滞留和其他生物学过程，如轴向模式和细胞周期调控（也是通过信号转导途径控制的）将是重要的。