Modification of Egg Plasma Membrane

University Of Kansas Medical Center, Kansas City KS

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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 kinases (PTKS) which function to initiate the calcium transient, as well as regulate pronuclear migration and fusion, initiation of DNA synthesis, and mitosis. Recent studies have demonstrated that the Fyn protein tyrosine kinase plays a major role in triggering the calcium transient that follows sperm-egg fusion. The objective of this proposal is to identify the mechanism by which Fyn activity is stimulated at fertilization. Using the sea urchin egg as a model system, we propose that Fyn kinase is activated by a tightly coupled phosphotyrosyl phosphatase which relieves Fyn from inhibition via dephosphorylation of Tyr 527. Specific aim I will characterize the phosphatase that acts on Fyn in the sea urchin egg. The role of this phosphatase in egg activation will be determined through microinjection experiments using mRNA encoding recombinant fusion proteins and blocking antibodies as specific inhibitors of phosphatase function. Specific aim II will characterize the sites of interaction between Fyn and this phosphatase. We will also determine the size of the c-Fyn pool that is activated at fertilization and determine if an amplification event occurs. Specific Aim III will test the hypothesis that c-Fyn is activated by a P-Tyr displacement mechanism at fertilization. Further studies will attempt to design a c-Fyn agonist based on the P-Tyr displacement mechanism The results of this study will identify the specific regulatory mechanism by which Fyn is activated at fertilization. This information will further the understanding of the biochemical mechanism by which the metabolic pathways and developmental programs in the egg are activated at fertilization and thereby enhance our ability to promote successful development in humans.