Quiescent Xenopus oocytes are activated by progesterone, which leads to resumption of the meiotic cell cycle and maturation of the oocyte into the egg. Progesterone binds to an unidentified and possibly novel membrane-associated receptor that is linked to a poorly understood signal transduction pathway that results in the translational activation of stored mos mRNA. Newly made mos protein, a MAP kinase kinase kinase, leads to activation of pre-MPF and progression through meiosis. The goal of this proposal is to identify the components of the progesterone-activated signaling pathway that lead to the translational recruitment of stored mos mRNA and cell cycle re-entry. 1. A pilot screen to find proteins that function early in the progesterone signaling pathway has identified two candidates, the ser/thr kinase Eg2 and the novel protein PTA10. Overexpression of Eg2 accelerates both the appearance of new mos protein and cell cycle re-entry, and makes ooycytes more sensitive to progesterone. We will use several different molecular approaches to determine exactly when Eg2 is activated, what kinase is responsible for Eg2 activation, what are the important targets of Eg2, and where Eg2 functions relative to other components in the pathway. 2. PTA10, which contains 5 WD40 repeats, motifs found in several signaling proteins, is phosphorylated soon after progesterone stimulation. The role of pTA10 in the progesterone signaling pathway will be determined. 3. The small pools cDNA screen for components of the signaling pathway will be continued to find additional components in the pathway, and the functional roles of these proteins will be determined. 4. We will continue to screen for novel progesterone-binding proteins, and test candidates for their ability to activate signaling in oocytes. In view of a recent report that, in certain mammalian cells, the conventional nuclear progesterone receptor can also activate rapid cytoplasmic signal transduction, we will also test the possibility that the conventional receptor functions as the signaling receptor in oocytes.
