PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes

Abstract

PTEN is one of the most frequent mutated tumor suppressor proteins in human cancer. This project investigates how PTEN interacts with lipid model membranes of different composition and morphology. We hypothesize that PTEN binds synergistically to the plasma membrane (PM) by interacting with phosphatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic lipids. While the currently accepted model is that PTEN's C2 domain interacts with phosphatidylserine (PS), we hypothesize that the C2 domain can interact with any anionic lipid, as long as this lipid has a sufficiently high local concentration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched domains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting that PI is a better second binding partner than PS. We are using stopped-flow fluorescence spectrophotometry as an ensemble technique that provides us with information about the association and dissociation kinetics of PTEN when interacting with lipid model membranes of different composition. Using this technique we have found that PTEN binds to pure PS and PI vesicles with a similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composition. From these data, we determine the PTEN diffusion coefficient, hopping frequency and dwell times. In the aggregate, this allows us to describe in detail PTEN binding mechanisms with differently composed lipid model membranes.