The goal of this project is to achieve a molecular understanding of the oncogenicity of phosphatidylinositol 3-kinase (PI3K). The proposed work will use two basic approaches: (1) a genetic analysis of the oncogenic functions of p110?, the catalytic subunit of PI3K, and (2) genome-wide interrogations for novel regulators and targets of PI3K. The genetic analysis will concentrate on the interactions between p110? and Ras as well as p110? and p85. These interactions are critical to the oncogenicity of p110?;they are fundamentally changed in the cancer-specific gain-of-function mutants of p110?. The p110? protein carrying cancer-specific mutations in the helical domain (E542K, E545K) has requirements for p85- and Ras-binding that are opposite to those of the kinase domain mutant (H1047R). The helical domain mutants depend on Ras-binding but are largely independent of p85-binding. The kinase domain mutant does not require Ras but needs p85 to be active. We plan to analyze mutants of p85 and of Ras in which specific functions are disabled for their ability to activate wild-type and mutant p110?. This genetic analysis will advance our understanding of the molecular mechanisms by which cancer-specific mutations in p110? induce a gain-of-function and make the protein oncogenic. The genome-wide interrogations for regulators and targets will take advantage of two new technologies. (1) A screen of expression libraries in the yeast Saccharomyces cerevisiae. This is a lethality screen based on the fact that expression of PI3K is toxic to Saccharomyces cerevisiae. The observed growth defect can be rescued by the expression of negative regulators of PI3K. Using this screen, we have identified several novel suppressors of PI3K. We will characterize these regulators and define the signaling chains that connect them to PI3K. (2) The second technology is a kinome screen that uses the two universally conserved lysines in ATP pockets of kinases to tag ATP-binding proteins by covalent linkage to biotin. We have used this method to reveal PI3K-induced changes in the kinome and, during initial studies, have identified three growth-regulatory kinases that are differentially expressed in PI3K- transformed cells. The roles of these and additional PI3K targets identified by the kinome screen will be analyzed and determined.
- Relevance The cancer-specific gain-of-function mutants of p110?, the catalytic subunit of PI3K, constitute the most promising therapeutic targets currently available. A complete understanding of these mutant proteins, their wild-type counterparts and their oncogenic actions is necessary to take advantage of this unique opportunity for developing novel, targeted cancer therapies.
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