An Optogenetic-Phosphoproteomic Approach Links Akt Activation to Dynamic Temporal Changes in Signaling in Endothelial Cells

The serine/threonine kinase Akt is a central node in cell signaling. While aberrant Akt activation underlies the development of a variety of human diseases, how different patterns of Akt-dependent phosphorylation dictates downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we performed a systems-level analysis that integrates method advances of optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different Akt intensity, durability, and pattern of stimulation activated temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identified a series of signaling circuits that define the sequential downstream cascade of Akt activation and interrogated how Akt signaling cross-talks with growth factor signaling in endothelial cells. Furthermore, our results categorized substrates of kinases that are preferably activated by oscillating, transient, and sustained Akt signals. We validated a list of Akt motif-carrying phosphosites that covaried with Akt phosphorylation across experimental conditions as potential Akt substrates and presented the entire dataset a rich resource for future studies on Akt signaling and dynamics.