Spatio-temporal Dynamics of Protein Kinase B/Akt Signaling Revealed by a Genetically Encoded Fluorescent Reporter

Abstract

The serine/threonine kinase protein kinase B (PKB)/ Akt is a critical regulator of insulin signaling, cell survival, and oncogenesis. The activation mechanisms of this key kinase are well characterized. In contrast, inactivation of PKB signaling by phosphatases is less well understood. To study the dynamics of PKB signaling in live cells, we generated a genetically encoded fluorescent reporter for PKB activity that reversibly responds to stimuli activating phosphatidylinositol 3-kinase. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer, allowing real-time imaging of phosphorylation catalyzed by PKB. Because of its reversibility, the reporter also allows termination of PKB signaling by phosphatases to be monitored. We found that PKB signaling in the cytosol was more rapid and more transient compared with that in the nucleus, suggesting the presence of differentially regulated phosphatase activity in these two compartments. Furthermore, targeting of the reporter to the plasma membrane, where PKB is activated, resulted in accelerated and prolonged response compared with the response in the cytosol, suggesting that release of PKB or its substrates from the membrane is required for desensitization of PKB signaling. These data reveal spatio-temporal gradients of both signal propagation and signal termination in PKB signaling. Protein kinase B (PKB)1 /Akt is a serine/threonine kinase that is the prominent mediator of pathways resulting in enhanced cell growth and cell survival. In 1991 it was identified as the transforming component of AKT8, a virus correlating with a high incidence of spontaneous lymphoma in mice, and named Akt (1). In the same year, two other groups isolated the same kinase via its homology to protein kinase A (PKA) and protein kinase C (PKC) and designated it Rac (related to the A and C kinases) (2) and PKB (3). There are three isozymes in mammals: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥. While the kinase is referred to by these two names, PKB and Akt, herein it will be referred to as PKB.PKB contains an NH 2 -terminal pleckstrin homology (PH) domain followed by a kinase domain and a short COOH-terminal regulatory tail containing an activating phosphorylation site (4). PKB is activated by recruitment to membranes following stimulation of growth factor receptors. Briefly, activated growth factor receptors lead to plasma membrane recruitment of phosphatidylinositol 3-kinase (PI 3-kinase), which leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) at the plasma membrane. The PH domain of PKB binds to the newly formed PIP 3 resulting in translocation of the kinase to the plasma membrane (5-8). Once at the membrane, PKB becomes activated via two sequential phosphorylation steps, first by its upstream kinase PDK-1 on Thr 308 within its activation loop, and next via autophosphorylation at Ser 473 within the COOH-terminal hydrophobic motif (9 -12). PKB is maximally active when phosphorylated at both regulat...

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II

“…In response to receptor-mediated PIP 3 formation, Akt translocates to the plasma membrane 34 where it is phosphorylated and activated. We demonstrate here that Akt activation by LIF/PI3K increases its association with the mitochondrial cell fraction.…”

“…10 A study using a FRET-based Akt activity reporter in NIH3T3 cells clearly shows that Akt activated at the plasma membrane translocates to other compartments including the nucleus. 34 It appears, therefore, that activated Akt can translocate to multiple cellular compartments, including mitochondria and the nucleus, to afford acute and chronic protective effects on cells.…”

“…To examine mitochondrial HK-II phosphorylation, the pellet was resuspended in RIPA buffer, HK-II immunoprecipitated, and the immunoprecipitate resuspended in kinase reaction buffer 34 containing 20 mM Hepes, 2 mM DTT and 5 mM MgCl 2 with or without 200 mM ATP and recombinant Akt (Upstate). Incubations were carried out at 301C for 15 min, spun down, resuspended in 2 Â LDS running buffer (Invitrogen) and boiled for 5 min.…”

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

“…Two types of FRET-based monitors of Akt activity have been reported previously (Calleja et al, 2003;Sasaki et al, 2003;Ananthanarayanan et al, 2005;Kunkel et al, 2005): 1) measurement of phosphorylation of Akt substrate (Sasaki et al, 2003;Kunkel et al, 2005) and 2) measurement of the conformational change of Akt (Calleja et al, 2003). Probes belonging to the first type detect the balance between the activities of Akt and antagonizing phosphatases.…”

Dynamic visualization of signal transduction in living cells: From second messengers to kinases

“…Such a modular design allows one to change only the substrate portion of the reporter to generate new biosensors that are specific for different kinases. For example, Bkinase activity reporter (BKAR) and D-kinase activity reporter (DKAR) were engineered by replacing the substrate domain in C-kinase activity reporter (CKAR) with consensus substrates for PKB and PKD, respectively (15,18).…”