The long term objective of this proposal is to identify the biochemical steps which act to transduce the initial stimulus of sperm-egg fusion into a signal to begin cell division. It is now well established that fertilization, like many other mitogenic events, involves activation of a tyrosine protein kinase and it has recently been shown that this results in phosphorylation, on tyrosine, of a high molecular weight egg cortex protein (HMWCP). This phosphorylation event is regulated by a fertilization-induced alkaline shift in pH, and occurs during the 'pH critical' stage of egg activation in which this alkaline shift is essential for successful induction of cell division. It is proposed that HMWCP phosphorylation acts as a pH sensitive transducer that responds to a relatively small change in pH, and, in turn, activates components of the cell cycle control which themselves are not sensitive to small changes in pH. This represents a novel signal transduction mechanism which amy function in a variety of other systems in which mitogenic stimuli induce an alkaline shift in cytoplasmic pH. The first specific aim of this proposal is to characterize the HMWCP immunohistochemically and biochemically in order to determine its subcellular location, relative phosphorylation state, and number of phosphorylation sites. Further biochemical analysis together with microinjection experiments will be done to examine the function of this protein and test the hypothesis that phosphorylation of the HMWCP plays a key role in egg activation. Specific attention will be paid to the possibility that HMWCP phosphorylation plays a role in triggering the cyclin/MPF pathway leading to initiation of mitosis.
The second aim will be to characterize the tyrosine kinase which phosphorylates the HMWCP and use an in vitro system to examine the mechanism for its sensitive regulation by pH.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Reproductive Biology Study Section (REB)
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University of Kansas
Schools of Medicine
Kansas City
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