Sensitive Akt biosensor and expansion microscopy reveal regulation of Akt at the lysosome

Abstract

The phosphoinositide 3-kinase (PI3K)/Akt pathway regulates a wide range of cellular processes, including cell survival, growth and metabolism. As a critical component of this pathway, the serine/threonine protein kinase Akt activates the downstream component mechanistic target of rapamycin complex 1 (mTORC1) through spatial control of the TSC complex at the lysosome but also has downstream targets throughout the cell. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, including the mechanism controlling its activity at the lysosome, are not clearly understood. To interrogate the regulation of Akt at subcellular locations, we developed a new genetically encoded fluorescent Akt activity biosensor. Coupling an Akt-specific substrate peptide and a phosphoamino acid binding domain to either side of circularly permutated GFP via optimized linkers yielded an excitation-ratiometric Akt activity reporter (ExRai-AktAR2) that exhibits excitation maxima at both ~400 nm and ~500 nm, along with a single emission peak near 515 nm, and undergoes an excitation ratio change of about 2 fold. Genetically targeted ExRai-AktAR2 revealed widespread Akt activity at different subcellular locations, including cytoplasm, plasma membrane, golgi, and nucleus. Using a lysosomally targeted ExRai-AktAR2, we showed that growth factor stimulation induces Akt activity at the lysosomal surface, which is dependent on presence of 3-phosphoinositides (3-PIs) on the lysosome membrane. Further dissection using pharmacological perturbation, genetically targeted manipulation of 3-PIs and expansion microscopy showed that lysosomal 3-PIs, which are dependent on dynamin-mediated endocytosis, are critical for recruiting Akt to the lysosome, resulting in accumulation of Akt activity and mTORC1 activity on the lysosomal surface. Thus, 3-PIs, a class of critical lipid second messengers that are mainly found in the plasma membrane, accumulate on lysosomal membrane to direct a multi-faceted kinase to organize lysosome-specific signaling for local activation of downstream effectors, showcasing intricate spatial organization and regulation of a key signaling pathway.