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.

mTORC2 plays critical roles in metabolism, cell survival, and actin cytoskeletal dynamics via phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae Dictyostelium cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling the supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by chemoattractant-induced phosphorylation of Rho-GDP at serine 192 by GSK-3. Furthermore, Rho-GDP rescued defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of serine 192. Thus, in contrast to the prevailing view that GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signaling is activated by Rho-GDP.

During chemotaxis, cells sense extracellular chemical gradients and position Ras GTPase activation and phosphatidylinositol (3,4,5)-triphosphate (PIP3) production toward chemoattractants. These two major signaling events are visualized by biosensors in a crescent-like zone at the plasma membrane. Here, we show that a Dictyostelium Rho GTPase, RacE, and a guanine nucleotide exchange factor, GxcT, stabilize the orientation of Ras activation and PIP3 production in response to chemoattractant gradients, and this regulation occurred independently of the actin cytoskeleton and cell polarity. Cells lacking RacE or GxcT fail to persistently direct Ras activation and PIP3 production toward chemoattractants, leading to lateral pseudopod extension and impaired chemotaxis. Constitutively active forms of RacE and human RhoA are located on the portion of the plasma membrane that faces lower concentrations of chemoattractants, opposite of PIP3 production. Mechanisms that control the localization of the constitutively active form of RacE require its effector domain, but not PIP3. Our findings reveal a critical role for Rho GTPases in positioning Ras activation and thereby establishing the accuracy of directional sensing.