Probing the Effects of Pathological Conditions on AKT

Project Summary The PI3K-AKT pathway is one of the most implicated pathways in disease. AKT is the main regulator of the pathway through its interactions with over one hundred protein partners to accomplish specific cellular outcomes including cell proliferation, survival, metabolism, and protein synthesis. To accomplish these cellular outcomes, AKT must first undergo a conformational change in the cytoplasm towards an active conformation, then associate at the plasma membrane, and finally interact with its binding partners. Despite its prominent role in major cellular functions and knowledge of AKT’s activation process, real time mechanistic information regarding how AKT becomes active, associates at the plasma membrane, and interacts with its binding partners under physiological and pathological conditions is not well understood. We hypothesize that biophysical differences in each of these steps of AKT’s activation process underlie pathway dysregulation in disease. Knowing this information is critical to understand the basic biology of the pathway under physiological and pathological conditions, characterize/reveal important kinetic vulnerabilities associated with AKT conformational bias/membrane association, and identify AKT-protein partner complexes that can be exploited in the long-term to treat disease. To interrogate this hypothesis, we have developed novel live-cell bioluminescent- and bioluminescence resonance energy transfer-based assays that will be applied to measure how 1) conformational changes and membrane association, and 2) populations of AKT-protein partner complexes vary between physiological and pathological conditions. These approaches overcome major limitations associated with studying purified protein and complex results from standard cell-based viability and signaling assays. The results of this proposal will be the first to reveal the kinetic differences in AKT activation as cells respond to pathological conditions, and can be applied to any system. The prosed work over the next five years will generate substantial data and represents a hypothesis generating platform to propel the field forward.