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 (PIP) or PI(3,4)P to the PH domain allosterically activates Akt by promoting high-affinity substrate binding. Conversely, dissociation from PIP 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 PIP 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.

mTORC2 integrates extracellular cues with pathways controlling growth and proliferation, but the spatial control of mTORC2 activity is unclear. Using a new reporter, Ebner et al. show that endogenous mTORC2 activity localizes to plasma membrane, mitochondrial, and endosomal pools, which display distinct sensitivity to growth factors.

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.

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. Recent studies using high-resolution imaging have demonstrated that signaling via TcR/CD3 displays complex spatial organization. TcR/CD3 molecules are preclustered at the plasma membrane and, upon ligation, nucleate downstream signaling components seconds after T-cell activation (4, 5). TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.Currently, phosphorylation of TcR/CD3 in cells and in vitro is monitored using phospho-specific antibodies, and there are no methods that enable dynamic monitoring of the spatial organization of CD3 phosphorylation in live cells. Here, we have constructed genetically encoded fluorescent reporters compatible with imaging of live and fixed cells and demonstrate that they accurately monitor the dynamics and intracellular organization of CD3ζ phosphorylation in Jurkat T cells. Using the reporters, we observed that in addition to the expected activationdependent phosphorylation at the plasma membrane, tyrosinephosphorylated CD3ζ accumulated in the perinuclear endosomal vesicles. Our results demonstrate that endosomal CD3ζ remains signaling-competent and suggest the possibility that internalized CD3ζ pool may help to sustain long-term signaling in T cells. Results Design and Characterization of the CD3ζ Phosphorylation Reporters.To generate a Förster's resonant energy transfer (FRET)-based monomolecular reporter for phosphorylation of the key CD3 signal-transducing subunit ζ, we fused the C terminus of CD3ζ to a pair of green and red fluorescent proteins, eGFP and mCherry, linked by a flexible spacer and followed by the tandem SH2 domains of human ZAP-70 (residues 1-259; tSH2 ). We reasoned that intramolecular binding of the SH2 domains to tyrosine-phosphorylated ITAMs of CD3ζ (7) would result in conformational rearrangement of the adjacent fluorescent proteins and change in the FRET efficiency between the dono...

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.

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 lipidcontrolled 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.

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.

Maintaining and modulating the mechanical anisotropy is essential for biological processes. How this is achieved on the microscopic scale in living soft matter is however not always clear. Here we introduce Brillouin Light Scattering Anisotropy Microscopy (BLAM) for mapping the high-frequency viscoelastic anisotropy inside living cells. Following proof-of-principle experiments on muscle myofibers, we apply this to study two fundamental biological processes. In plant cell walls we show how a phase-transition driven switch between anisotropic-isotropic wall properties may lead to asymmetric growth. In mammalian cell nuclei we uncover a spatio-temporally oscillating elastic anisotropy correlated to chromatin condensation, with long range orientational correlations that may provide a dynamic framework for coordinating intra-nuclear processes. Our results highlight the direct and indirect role the high-frequency mechanics can play in providing dynamic structure that lead to the regulation of diverse fundamental processes in biological systems, and offer a means for studying these. BLAM should find diverse biomedical and material characterization applications.