MODIFICATION OF EGG PLASMA MEMBRANE

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DESCRIPTION (Adapted from the Investigator's Abstract): The long term objective of this proposal is to identify the biochemical steps which transduce the initial signal of sperm-egg fusion into the various responses of the egg to fertilization. It is now established that egg activation involves the action of protein tyrosine kineses (PTKs) which function to regulate pronuclear migration and fusion, initiation of DNA synthesis, and mitosis. In addition, recent studies have demonstrated that protein tyrosine kinase activity plays a unique and essential role in the fertilized egg to program developmental events at the gastrula stage. The objective of this proposal is to identify those processes in the egg that are regulated by the fertilization induced activation of Fyn kinase activity and to determine the mechanism by which they are controlled. Using the sea urchin egg as a model system, the PI proposes that Fyn kinase functions downstream of the initial signal for egg activation, acting to regulate one or more specific PTK-dependent functions which may include pronuclear migration, DNA synthesis, and mitosis. Specific aim I will characterize the egg isoform of Fyn kinase and study its expression in the egg. The role of Fyn kinase in egg activation will be determined through microinjection experiments using recombinant GST fusion proteins encoding the unique SH2 and SH3 domains of Fyn N-terminal as specific inhibitors of Fyn function. The hypothesis that Fyn stimulates DNA synthesis via RAS activation will be tested. Specific aim II will identify the protein substrates of Fyn kinase in the egg. This will allow the PI to establish the specific biochemical pathways that are activated in response to Fyn activity. Specific Aim III will study the mechanism by which Fyn kinase activity is stimulated at fertilization. Particular emphasis will be placed on the effect of other protein kinases and phosphatases in regulating Fyn catalytic activity.