Activation of PI3K by Ras: Single Molecule Studies of the Activation Mechanism

The lipid-anchored GTPase Ras is well known for its role in oncogenesis – about one quarter of all human tumors show mutations in a Ras family member. One of the best characterized Ras targets is the lipid kinase Phosphatidylinositol 3-Kinase (PI3K) which, like Ras, is highly oncogenic. PI3K phosphorylates the constitutive plasma membrane lipid phosphatidyl-inositol-(4,5)-bisphosphate (PIP2) yielding the essential signaling lipid phosphatidylinositol-(3,4,5)-bisphosphate (PIP3). The resulting PIP3 lipid signal regulates an array of cell processes including migration and growth.The underlying mechanism by which Ras activates PI3K is unclear - this project aims to better elucidate the activation mechanism using single molecule TIRF microscopy to probe the interactions and activities of single proteins on a target membrane surface. One hypothesis is that the lipid-anchored, membrane-bound Ras protein increases the surface density of PI3K on the membrane by increasing kon and/or decreasing koff. An alternative hypothesis proposes that Ras binding increases the PI3K kcat and/or affinity for substrate lipid PI(4,5)P2 (PIP2) and/or ATP. Single-molecule fluorescence methods will be used to distinguish between these possibilities by observing the number of single particle diffusion tracks, track length, diffusion speed, specific activity, kcat, and Km of PI3K in the presence and absence of Ras. Our poster will present the latest findings of these studies.