The objective of this project is to determine the role of the egg plasma membrane in transducing the initial stimulus of sperm-egg fusion into the various metabolic responses that occur as a consequence of fertilization. Using the sea urchin fertilization system, we will study the role of a plasma membrane bound tyrosine specific protein kinase in the fertilization process. The first priority will be to monitor the activity of the tyrosine specific protein kinase during fertilization and determine what events (Cortical reaction, pH elevation, initiation of protein synthesis) regulate its activity. We will then identify the protein substrates that are phosphorylated by this enzyme at fertilization and localize the enzyme and its substrates by subcellular fractionation techniques. Finally, we will purify and characterize the enzyme so that it can be compared to tyrosine specific protein kinases in other systems. Monoclonal antibodies will be raised against the partially purified kinase and screened for their ability to stimulate or inhibit kinase activity. These antibodies will then be microinjected into eggs to study the effect of elevated or decreased tyrosine phosphorylation on development. Finally, we will attempt to measure tyrosine specific kinase activity and identify phosphotyrosine containing proteins in the mouse oocyte and 2 cell embryo. In this way, we will confirm that the major findings obtained in the sea urchin system have relevance to mammalian development. The results of these studies will provide new information on the mechanism by which egg metabolism and cell division are regulated at fertilization. This information may also find application in other development systems where the regulation of cell growth is involved.
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