Activation of phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine kinase Akt (also known as protein kinase B) triggers a cascade of responses that are critical for tumorigenesis, from cell growth and proliferation to survival and mobility. Aberrations of components in the PI3K/Akt pathway have been shown to be present in a majority of tumors. We hypothesize that aberrant PI3K/Akt activation could be characterized by combined activity profiles and used as a diagnostic marker in cellular activity-profiling. To test this hypothesis, we propose the following specific aims: 1) To analyze the activities of PI3K and Akt in breast cancer cell lines and to further develop fluorescent activity sensors for various components in the PI3K/Akt pathway; 2) To develop cellular assay platforms for high throughput activity-profiling of oncogenic PI3K/Akt signaling. These studies will take advantage of a series of fluorescence resonance energy transfer (FRET)-based reporters we have recently developed for measuring the activities of Akt and PI3K in living mammalian cells. Fluorescent activity sensors and cellular assay platforms developed in this study can be used in systematic analysis of the critical components in PI3K/Akt pathway in various cancers to generate activity profiles. Correlation of genetic alterations with activity profiles and phenotypes should provide new insights into the molecular mechanisms of cancer development. On the other hand, molecular diagnostics based on such activity-profiling could identify the molecular defects and the malfunctioned key nodes in the signaling network for a given cancer, and guide appropriate molecular therapeutics as well as facilitate their development and evaluation. ? ? ?
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