GENETIC MUTATIONS OF PROTEIN KINASE INHIBITOR

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DESCRIPTION: A vast array of hormones, upon binding to their cognate receptors on the cell surface, cause an increase in intracellular cAMP, the principal target of which is cAMP-dependent protein kinase (PKA). This system constitutes a major signaling pathway by which animal physiology is regulated. The Walsh inhibitor, or PKI, is a potent and highly specific inhibitor of PKA that was discovered nearly 30 years ago in skeletal muscle. A second inhibitor, referred to as testis PKI, was subsequently identified. It is now known that these two isoforms of PKI are encoded by unique genes, and are expressed in a distinct, but overlapping, assembly of tissues. Importantly, both are expressed in testis, where they are differentially regulated. Despite extensive biochemical characterization, including the recent discovery that PKI exhibits a nuclear export activity, little is known about the physiological role of PKI. The goal of this project is to examine the importance of the inhibitory and nuclear export activities of PKI in male reproductive function. This project uses embryonic stem cell mediated gene targeting technology to develop mouse strains carrying either an inactivating mutation in each PKI gene or a subtle mutation that inactivates the nuclear export signal of PKI. The phenotypic and biochemical outcome of these mutations will be assessed. Preliminary studies will identify which of the cell types (germ cells vs. non-germ cells) of the testis express the two PKI isoforms, and will provide the basis for subsequent analysis of the mutant mouse lines. In addition, the nuclear export signal of PKI will be identified by transient transfection studies in cultured cells in preparation for the gene targeting in mice. These studies will identify defects in testicular function resulting from a loss of, or a functional alteration in PKI, as well as provide PKI-deficient mice for future studies on the role of PKI in non- reproductive tissues. It is anticipated that these molecular genetic approaches will provide a wealth of novel information about the physiological function of PKI, and will contribute significantly to our overall understanding of the cAMP second messenger system in male reproduction and fertility, an area of global concern.