Mechanism of PTEN binding to model membranes

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a potent tumor suppressor. PTEN’s tumor suppressor action is rooted in its phosphatase function on the lipid substrate phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). PTEN’s enzymatic activity is specific for the third position of the inositol headgroup. PI(3,4,5)P3 is a second messenger that is a part of the PI3K-Akt pathway, and its dysregulation leads to constitutively activated AKT. The result of AKT activation is cell cycle progression, motility, cell growth, and proliferation, and consequently, overaction leads to neoplastic growth and tumorigenesis. PTEN antagonizes this pathway by regulating PI(3,4,5)P3 population through its phosphatase activity which produces the lipid PI(4,5)P2 (phosphatidylinositol-(4,5)-bisphosphate). A result of PTEN’s function is that its activity must be localized at the PM (plasma membrane) since this is where its substrate resides. Additionally, the mole percent of the phosphoinositide family of lipids is small. From highest percent composition to lowest the phosphoinositide species in the PM rank as PI(4,5)P2 (~2%), PI(4)P (~1%), and PI(3,4,5)P3 (~0.02%). For PTEN to turn over its substrate, it must first translocate from the cytosol to the PM and then search through the plasma membrane for this rare but high in demand lipid. This is at the center of the scarcity paradox. This work explores how PTEN may overcome this paradox by using its multiple lipid binding domains to interact with multiple lipid partners to efficiently localize it toward a region with a high probability of having PI(3,4,5)P3. This hypothesis is tested using two kinetic methodologies. First, we use pre- steady state stopped-flow spectrometry to determine the rates that govern PTEN-lipid binding. Second, we use single-molecule total internal reflectance fluorescence (smTIRF) microscopy to resolve the diffusion coefficients and dwell times of bound PTEN on SLBs supported lipid bilayers (SLBs). We test PTEN against various lipid compositions to determine how the bilayer structure in addition to the chemistry of the lipid influences the enzyme’s binding. These compositions include PI(4,5)P2, PI phosphatidylinositol (PI), phosphatidylserine (PS), PI(4,5)P2/PI and PI(4,5)P2/PS. In addition to this kinetic work, we will also present a novel model membrane platform that takes advantage of a microfluidic device to develop lateral lipid gradients in SLBs. This microfluidic platform, in the future, will allow for the investigation of the dynamic behavior of proteins interacting with lipids but with a bilayer that has a structure recapitulating polarized membranes like in chemotaxing cells