Phosphatase and tensin homolog (PTEN) is one of the most frequently mutated tumor suppressor genes in cancers. PTEN plays a central role in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling and converts PIP3 to phosphatidylinositol (4,5)-bisphosphate (PIP2) at the plasma membrane. Despite its importance, the mechanism that mediates membrane localization of PTEN is poorly understood. Here, we generated a library that contains GFP fused to randomly mutated human PTEN and expressed the library in Dictyostelium cells. Using live cell imaging, we identified mutations that enhance the association of PTEN with the plasma membrane. These mutations were located in four separate regions, including the phosphatase catalytic site, the calcium-binding region 3 (CBR3) loop, the Cα2 loop and the C-terminal tail phosphorylation site. The phosphatase catalytic site, the CBR3 loop and the Cα2 loop formed the membrane-binding regulatory interface and interacted with the inhibitory phosphorylated C-terminal tail. Furthermore, we showed that membrane recruitment of PTEN is required for PTEN function in cells. Thus, heterologous expression system in Dictyostelium cells provides mechanistic and functional insight into membrane localization of PTEN.

The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a key regulator of cell proliferation, survival, and migration and the enzyme that dephosphorylates it, phosphatase and tensin homolog (PTEN), is an important tumor suppressor. As excess PIP3 signaling is a hallmark of many cancers, its suppression through activation of PTEN is a potential cancer intervention. Using a heterologous expression system in which human PTEN-GFP is expressed in Dictyostelium cells, we identified mutations in the membrane-binding regulatory interface that increase the recruitment of PTEN to the plasma membrane due to enhanced association with PI(4,5)P2. We engineered these into an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. Upon expression in human cells, ePTEN decreases PIP3 levels in the plasma membrane; phosphorylation of AKT, a major downstream event in PIP3 signaling; and cell proliferation and migration. Thus, the activation of PTEN can readjust PIP3 signaling and may serve as a feasible target for anticancer therapies.protein engineering | PI3 kinase signaling | membrane localization | chemotaxis | protein interaction