A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity

Abstract

The serine/threonine protein kinase Akt regulates a wide range of cellular functions via phosphorylation of various substrates distributed throughout the cell, including at the plasma membrane and endomembrane compartments. Disruption of compartmentalized Akt signaling underlies the pathology of many diseases such as cancer and diabetes. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, particularly the mechanism controlling its activity at the lysosome, are not clearly understood. We developed a highly sensitive excitation-ratiometric Akt activity reporter (ExRai-AktAR2), enabling the capture of minute changes in Akt activity dynamics at subcellular compartments. In conjunction with super-resolution expansion microscopy, we found that growth factor stimulation leads to increased colocalization of Akt with lysosomes and accumulation of lysosomal Akt activity. We further showed that 3-phosphoinositides (3-PIs) accumulate on the lysosomal surface, in a manner dependent on dynamin-mediated endocytosis. Importantly, lysosomal 3-PIs are needed for growth-factor-induced activities of Akt and mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface, as targeted depletion of 3-PIs has detrimental effects. Thus, 3-PIs, a class of critical lipid second messengers that are typically found in the plasma membrane, unexpectedly accumulate on the lysosomal membrane in response to growth factor stimulation, to direct the multifaceted kinase Akt to organize lysosome-specific signaling.

Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

“…This allowed us to clearly detect nuclear AMPK activity, which has posed a challenge for FRET-based AMPKARs 16 , 17 . Future engineering efforts will focus on enhancing the dynamic range of some of the subcellularly targeted variants, as mito-ExRai AMPKAR exhibited a reduced dynamic range compared to an untargeted counterpart, presumably due to close proximity with membranes, as we have seen previously with other targeted biosensors 16 , 57 . We found that switching the reporting unit could affect the kinetics of the response (Fig.…”

Protocol for reading and imaging live-cell PKA activity using ExRai-AKAR2

“…Measured as the ratio of fluorescence intensities at these two excitation wavelengths, the PKA-stimulated response from ExRai-AKAR is up to 3-fold higher than optimized FRET-based AKARs, making ExRai-AKAR the most sensitive kinase sensor at the time. Much like the original AKAR spawned a large family of FRET-based KARs, the generalizable design of ExRai-AKAR has also yielded a series of highly sensitive reporters for other kinases, such as PKC and Akt ( Chen et al, 2021 , Mehta et al., 2018 ). At the same time, we realized that ExRai-AKAR can be further optimized through directed evolution by targeting its short linker regions, given the importance of linker sequences in biosensor engineering ( Akerboom et al., 2012 ; Dana et al., 2019 ; Miyawaki et al., 1997 ; Hires et al., 2008 ; Nakai et al., 2001 ; Patriarchi et al., 2018 ).…”

AktAR and Akt‐STOPS: Genetically Encodable Molecular Tools to Visualize and Perturb Akt Kinase Activity at Different Subcellular Locations in Living Cells

“…The following protocol describes the use of the FRET-based Akt activity reporter 2 (Ak-tAR2) and Akt-STOPS that are genetically targeted to specific subcellular locations to track and perturb, respectively, Akt activity in NIH3T3 cells, in response to PDGF stimulation (Chen, Sun, Zhong, Zhou, & Zhang, 2021;. Briefly, NIH3T3 cells are transfected with plasmid DNA encoding AktAR2 either alone, to assess cellular Akt activity induced by growth factors, or in combination with Akt-STOPS, to evaluate the inhibition of Akt activity by Akt-STOPS.…”

“…Furthermore, an allosteric lipid switch model for cellular Akt activation has been proposed, which suggested that phosphorylated, active Akt is restricted to 3‐PI‐containing membranes, and association with 3‐PI prevents the dephosphorylation and deactivation of Akt (Ebner, Lucic, Leonard, & Yudushkin, 2017; Yudushkin, 2020). Using subcellularly targeted AktARs, we detected Akt activity at various membrane compartments (Chen et al., 2021; Gao & Zhang, 2008; Zhou et al., 2020). In particular, 3‐PIs, which predominantly accumulate at the plasma membrane, unexpectedly accumulate at the lysosomal membrane in response to growth factor stimulation, specifically promoting lysosomal Akt/mTOR signaling (Chen et al., 2021).…”

“…Using subcellularly targeted AktARs, we detected Akt activity at various membrane compartments (Chen et al., 2021; Gao & Zhang, 2008; Zhou et al., 2020). In particular, 3‐PIs, which predominantly accumulate at the plasma membrane, unexpectedly accumulate at the lysosomal membrane in response to growth factor stimulation, specifically promoting lysosomal Akt/mTOR signaling (Chen et al., 2021). However, robust Akt activity is also detected in the cytosol and in the nucleus (Chen et al., 2021; Zhou et al., 2020), in addition to the plasma membrane and endomembrane surfaces.…”