Engineering ePTEN,an enhanced PTEN with increased tumor suppressor activities

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.Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a potent second messenger that drives many biological processes, such as cell growth, survival, and migration (1, 2). In many cancers, PIP3 levels are elevated due to mutations that either elevate the activity of phosphoinositide 3-kinases (PI3Ks) or decrease that of tumor suppressor phosphatase and tensin homolog (PTEN) (3–5). Although inhibition of PI3Ks has been extensively tried as a cancer drug target, activation of PTEN has been rarely studied (6). As PTEN is mainly located in the cytosol and its PIP3 phosphatase activity is suppressed at this location (7, 8), recruiting more PTEN to the plasma membrane and thereby stimulating its lipid phosphatase activity would seem to be an effective method to repress abnormal PIP3 levels in cancer cells.PTEN comprises an N-terminal “PIP2-binding” motif, globular catalytic and C2 domains, and a C-terminal tail (8–10). Positively charged residues in the PIP2-binding and C2 domains have been proposed to recruit PTEN to the plasma membrane through associations with negatively charged head groups of membrane lipids (11–13). The C-terminal tail is thought to fold back and bind to the membrane-binding regions, maintaining the majority of PTEN in the cytoplasm (11, 14, 15). This intramolecular inhibition is controlled by phosphorylation of four serine/threonine residues in the tail domain. A PTEN mutant that carries an alanine substitution in the phosphorylation sites of the C-terminal tail (termed PTEN_A4) increases the membrane association of PTEN. However, most PTEN_A4 is still present in the cytoplasm, suggesting that the A4 mutations may not completely liberate the membrane-binding sites from inhibition by the tail.To decipher the mechanisms underlying the membrane association of PTEN, we developed a visual screen for the localization of human PTEN expressed in Dictyostelium cells. PTEN is evolutionarily conserved, and human PTEN can functionally replace Dictyostelium PTEN (16–19). Using this heterologous expression system, we identified a membrane-binding regulatory interface in PTEN, consisting of regions of the catalytic domain and the CBR3 and Cα2 loops of the C2 domain (20). In the current study, we introduce multiple mutations in the membrane-binding regulatory interface that completely release the inhibitory effects of the tail, generating a synthetic enzyme, referred to as enhanced PTEN (ePTEN), with greatly increased membrane localization and PIP3 phosphatase activity. Our findings demonstrate that activation of PTEN is a feasible therapeutic strategy for cancers with increased PIP3 signaling.