The Akt protooncoprotein, also known as PKB, contains an aminoterminal PH domain and a carboxyterminal kinase domain, related to the kinase domains of PKA and PKC. Akt belongs to a family of kinases that includes at least three members in mammals and is conserved in eukaryotes from Dictyostelium to man. Studies by the PI and others over the last four years have shown that the Akt kinase is activated by growth factors via a multiple step process that includes binding of D3 phosphorylated phosphoinositides to the PH domain of the kinase, followed by its membrane translocation and phosphorylation of T308 and S473 by at least two separate kinases. As a target of the PI-3 kinase, Akt is required for the transduction of a variety of PI-3 kinase-generated signals including those that inhibit apoptosis and promote oncogenesis. Regarding the mechanism of Akt activation, the PI has shown that it interacts with the cdc37/Hsp90 complex. In addition, the PI has identified a novel Akt activation pathway that depends on the synergistic action of cytoplasmic tyrosine kinases and small GTPases. Regarding the function of the activated Akt, Dr. Tsichlis has recently shown that Akt PH domain mutants activated by the addition of an aminoterminal myristylation signal are transformation defective and exhibit weak anti-apoptotic activity in culture, suggesting that the Akt PH domain does not only regulate the process of Akt activation, but also contributes to the selection of Akt targets. Such targets may include several PH-domain binding proteins he has isolated using a two hybrid screen using the Akt PH domain as a bait. Additionally, he has shown that Akt contributes to the activation of NF-kB, induces expression of beta-catenin and interacts with the novel adapter protein Tvl-1 that is involved in the regulation of apoptosis. Moreover, using recently established transgenic mice expressing wildtype and mutant Akt proteins in the thymus, the PI has shown that Akt may contribute to the regulation of thymocyte positive and negative selection and thymocyte differentiation in vivo. Pertinent to both Akt activation and function is a novel yeast based genetic strategy that he has established that permits the identification of novel Akt regulators and targets. The proposed studies will utilize molecular genetics, tissue culture and transgenic mouse technologies to explore these novel observations in order to better redefine the role of Akt in signal transduction and oncogenesis.
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