Phosphatase and tensin homolog (PTEN) is one of the most frequently mutated tumor suppressor genes in cancers. PTEN has a central role in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling and converts PIP3 to phosphatidylinositol (4,5)-bisphosphate 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 green fluorescent protein 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.
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