Recent work from this laboratory provide evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related to the histidine protein kinases previously thought to be present only in prokaryotic cells. A member of this family corresponding to the pyruvate dehydrogenase (PDH) kinase, responsible for phosphorylation and inactivation of the mitochondrial PDH complex, has been cloned. A high titer antiserum against a recombinant PDH kinase fusion protein has also been generated. Previous work indicates that long term regulatory mechanisms are involved in control of PDH kinase activity, and, therefore, the proportion of the PDH complex in the active, dephosphorylated state. Mechanisms responsible for regulation of PDH activity are important because of the central role of this complex in carbohydrate metabolism. Our working hypothesis is that isozyme of PDH kinase exist and that long-term control mechanisms differentially affect the amounts and the specific activities of the isozyme. The hypothesis will be tested in experiments proposed with purified enzymes, recombinant enzymes, and tissues obtained from rats and humans.
The specific aims are to determine the molecular basis for (a) the presence of free and bound forms of PDH kinase; (b) the differences in the relative amounts of PDH kinase activity in liver, heart, kidney, muscle, brain, adipose tissue and tumor cells; and (c) changes in the relative amounts of PDH kinase activity induced by starvation and diabetes in the rat and by type II diabetes in man. The proposed work will contribute significantly to our understanding of mechanisms regulating fuel selection in mitochondria-containing tissues of the body. We should discover why PDH kinase activity increases in tissues of starved and diabetic animals, andy why the PDH complex is resistant to regulation by covalent modification in tumor cells. Since PDH kinase is a possible target for therapeutic intervention, the proposed work is relevant to the development of strategies for the treatment of diabetes, obesity, atherosclerosis, sepsis, and cancer.
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