Cytoplasmic Retention as a Regulatory Mechanism in Embryogenesis

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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a 670kD Drotein complex consisting of xnf7 and two other proteins (Shou et al., 1996; Kuang, Che, Elhodiri and Etkin, unpublished).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.