Project Summary PTEN, a key member of the PI3K pathway, exerts its main effect as a tumor suppressor by directly antagonizing the activity of PI3K as an interfacial lipid phosphatase, via dephosphorylating membranous PIP3, resulting in lower AKT activation. Expression of PTEN is lost in many cancer types, including breast, prostate and glioblastoma, resulting in heightened AKT activity, which favors cell growth. Recent work in the Parsons lab led to the discovery of PTEN-L, a secreted PTEN translational isoform, which can re-enter cells and dephosphorylate PIP3 in the recipient cells. Previous studies from the Parsons group and others have indicated that exogenous PTEN-L can enter tumor cells in PTEN null xenograft models and cause tumor regression and attenuation of PI3K signaling. These works highlight the great potential for PTEN-L to be used as a targeted therapy for patients with tumors bearing loss of PTEN. Although PTEN-L shares all domains of PTEN, including the phosphatase domain, C2 domains, and the C-tail, PTEN-L also has an additional 173 amino acids at the N-terminus, the function of which is still under investigation. Both PTEN and PTEN-L can be found in the cytoplasm and at membranes but must be recruited to membranes in order to dephosphorylate PIP3. Published work indicates that the N-terminal extension of PTEN-L, namely the membrane binding helix (MBH) domain, causes increased affinity for the membrane with partially diminished phosphatase activity. The goal of this project is to study the interfacial catalysis of PTEN-L in order to determine which PTEN-L domains are required for its phosphatase activity against membranous PIP3 and determine the success of purified PTEN-L with alterations to the native domains in treating tumors with aberrant PI3K signaling. In the first aim, we will determine the domains of PTEN- L that are important for membrane localization and its subsequent lipid phosphatase activity; both functions are vital for dephosphorylation of membranous PIP3. To test the requirement of the domains, we will mutate the membrane localization domains of PTEN-L. We will test the effects of these mutants in PTEN null cells on downstream PI3K signaling and localization to endogenous membranes in vitro and using biochemical assays. In the second aim, we will determine whether exogenous PTEN-L domain mutant proteins can be effective in treating cancer with aberrant PI3K signaling and whether these alterations to PTEN-L domains will improve therapeutic efficacy. We will first test the ability of the domain mutants to suppress cell growth in PTEN null cells in vitro. We will determine efficacy of purified PTEN-L domain mutants that retain growth suppression by measuring downstream PI3K signaling and growth rates of treated PTEN null cell lines, and through the use of xenograft and allograft models of PTEN null cancers. Next, we will determine whether any tested exogenous PTEN-L mutant proteins have favorable pharmacokinetics, ...