Control of Akt activity and substrate phosphorylation in cells.

Protein kinase B/Akt is a serine/threonine kinase that links receptors coupled to the PI3K lipid kinase to cellular anabolic pathways. Its activity in cells is controlled by reversible phosphorylation and an intramolecular lipid-controlled allosteric switch. In this review, I outline the current progress in understanding Akt regulatory mechanisms, define three models of Akt activation in cells, and highlight how intramolecular allosterism cooperates with cell-autonomous mechanisms to control Akt localization and activity and direct it toward specific sets of substrates in cells.

Getting the Akt Together: Guiding Intracellular Akt Activity by PI3K.

Intracellular signaling pathways mediate the rapid response of cells to environmental cues. To control the fidelity of these responses, cells coordinate the activities of signaling enzymes with the strength, timing, and localization of the upstream stimuli. Protein kinase Akt links the PI3K-coupled receptors to cellular anabolic processes by phosphorylating multiple substrates. How the cells ensure that Akt activity remains proportional to upstream signals and control its substrate specificity is unclear. In this review, I examine how cell-autonomous and intrinsic allosteric mechanisms cooperate to ensure localized, context-specific signaling in the PI3K/Akt axis.

TAPPing into PIPs: A new reporter reveals the origin of plasma membrane PI(3,4)P2.

The membrane lipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) is an important signaling effector, controlling both anabolic pathways and membrane trafficking. In this issue, Goulden et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201809026) report a new PI(3,4)P2 probe and show that plasma membrane PI(3,4)P2 is a product of PI(3,4,5)P3 dephosphorylation.

Localization of mTORC2 activity inside cells.

Activation of protein kinase Akt via its direct phosphorylation by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) couples extracellular growth and survival cues with pathways controlling cell growth and proliferation, yet how growth factors target the activity of mTORC2 toward Akt is unknown. In this study, we examine the localization of the obligate mTORC2 component, mSin1, inside cells and report the development of a reporter to examine intracellular localization and regulation by growth factors of the endogenous mTORC2 activity. Using a combination of imaging and biochemical approaches, we demonstrate that inside cells, mTORC2 activity localizes to the plasma membrane, mitochondria, and a subpopulation of endosomal vesicles. We show that unlike the endosomal pool, the activity and localization of mTORC2 via the Sin1 pleckstrin homology domain at the plasma membrane is PI3K and growth factor independent. Furthermore, we show that membrane recruitment is sufficient for Akt phosphorylation in response to growth factors. Our results indicate the existence of spatially separated mTORC2 populations with distinct sensitivity to PI3K inside cells and suggest that intracellular localization could contribute to regulation of mTORC2 activity toward Akt.

PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes.

Protein kinase B/Akt regulates cellular metabolism, survival, and proliferation in response to hormones and growth factors. Hyperactivation of Akt is frequently observed in cancer, while Akt inactivation is associated with severe diabetes. Here, we investigated the molecular and cellular mechanisms that maintain Akt activity proportional to the activating stimulus. We show that binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3) or PI(3,4)P2 to the PH domain allosterically activates Akt by promoting high-affinity substrate binding. Conversely, dissociation from PIP3 was rate limiting for Akt dephosphorylation, dependent on the presence of the PH domain. In cells, active Akt associated primarily with cellular membranes. In contrast, a transforming mutation that uncouples kinase activation from PIP3 resulted in the accumulation of hyperphosphorylated, active Akt in the cytosol. Our results suggest that intramolecular allosteric and cellular mechanisms cooperate to restrict Akt activity to cellular membranes, thereby enhancing the fidelity of Akt signaling and the specificity of downstream substrate phosphorylation.

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment.

Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/CD3 signaling after the receptor internalization.

Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B.

Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on Förster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells. We observed the establishment of a steady-state ES gradient across the cell. This gradient exhibited robustness to cell-to-cell variability, growth factor activation, and RTK localization, which demonstrated spatial regulation of PTP1B activity. Such regulation may be important for generating distinct cellular environments that permit RTK signal transduction and that mediate its eventual termination.