Optical Control of Phosphoinositide Binding: Rapid Activation of Subcellular Protein Translocation and Cell Signaling

• Published in: ACS synthetic biology 2021-11, Vol.10 (11), p.2886-2895
• Main Authors: Ryan, Amy, Hammond, Gerald R. V, Deiters, Alexander
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Cell Communication - physiology; Cell Line; Cell Membrane - metabolism; Cell Membrane - physiology; Green Fluorescent Proteins - metabolism; MAP Kinase Signaling System - physiology; Mice; Phosphatidylinositol 3-Kinases - metabolism; Phosphatidylinositols - metabolism; Phospholipase C delta - metabolism; Protein Binding - physiology; Protein Transport - physiology; Signal Transduction - physiology; Sorting Nexins - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.1c00328

Cells utilize protein translocation to specific compartments for spatial and temporal regulation of protein activity, in particular in the context of signaling processes. Protein recognition and binding to various subcellular membranes is mediated by a network of phosphatidylinositol phosphate (PIP) species bearing one or multiple phosphate moieties on the polar inositol head. Here, we report a new, highly efficient method for optical control of protein localization through the site-specific incorporation of a photocaged amino acid for steric and electrostatic disruption of inositol phosphate recognition and binding. We demonstrate general applicability of the approach by photocaging two unrelated proteins, sorting nexin 3 (SNX3) and the pleckstrin homology (PH) domain of phospholipase C delta 1 (PLCδ1), with two distinct PIP binding domains and distinct subcellular localizations. We have established the applicability of this methodology through its application to Son of Sevenless 2 (SOS2), a signaling protein involved in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Upon fusing the photocaged plasma membrane-targeted construct PH–enhanced green fluorescent protein (EGFP), to the catalytic domain of SOS2, we demonstrated light-induced membrane localization of the construct resulting in fast and extensive activation of the ERK signaling pathway in NIH 3T3 cells. This approach can be readily extended to other proteins, with minimal protein engineering, and provides a method for acute optical control of protein translocation with rapid and complete activation.