Cytoplasmic Retention as a Regulatory Mechanism in Embryogenesis

细胞质保留是控制核蛋白功能的重要调控步骤。它是一种普遍现象，发生在从酵母到人类的各种系统中，并在细胞周期、不同分化阶段和发育阶段调节蛋白质的合成。在发育过程中，果蝇（Govind and Steward, 1992）和爪蟾（Dreyer et al., 1983; Miller et al., 1989,1991）的细胞质中存在背侧基因产物的保留。Etkin博士正在分析一种晚期转移蛋白，Xenopus核因子7 (xnf7)，它起源于卵母细胞核（GV），并保留在细胞质中，直到它在囊胚中期转移（MBT）时进入细胞核（Miller et al., 1989,1991; Reddy et al.,1991）。Xnf7是一个由原基因和转录因子组成的锌指基因家族的成员（Ready and Etkin, 1991; Reddy et al., 1992; Freemont, 1993），最近被证明在胚胎的背腹侧模式中起作用（El-Hodiri et al.，提交）。Li等人（1994a）证明存在一个22个氨基酸的细胞质保留域（CRD），它需要两个磷酸化位点（位点1和2）的磷酸化才能在xnf7的细胞质保留中发挥作用（Shou等人，1996）。Etkin博士还表明，xnf7的细胞质保留很可能涉及锚定机制（Li et al.,1994a），并且该蛋白存在于由xnf7和其他两种蛋白组成的670kD蛋白质复合物中（Shou et al., 1996; Kuang, Che， Elhodiri和Etkin，未发表）。他的假设是，在MBT之前，xnf7在细胞质中的保留是位点1和位点2被特定激酶磷酸化的结果，这些激酶影响了CRD与细胞质锚定复合物的相互作用。在本提案中，他概述了一系列旨在验证这一假设的实验方法，并通过解决以下具体目标来描述锚复合物的组成部分：鉴定使调节细胞质保留的位点1和2磷酸化的激酶；2. 利用生物化学和遗传学方法鉴定xnf7相互作用蛋白（XIP）；3.对XIP蛋白进行功能分析。他认为，使用xnf7作为模型对这种现象进行分析，将成为理解许多系统中细胞质保留机制基本原理的范例。此外，鉴定调节CRD保留功能的蛋白激酶将揭示调节这一过程的信号转导途径的性质。这对于将xnf7的细胞质保留与其他生物过程（如轴向模式和细胞周期调节）整合起来非常重要，这些过程也通过信号转导途径控制。

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.