Optogenetic Control of Phosphatase and Kinase Function

Abstract

Signal transduction is an essential mechanism of cellular function. These pathways are responsible for eliciting a physiological response to external stimuli, resulting in biological processes such as cell cycle progression, proliferation, and cell fate determination. However, the complex networks of protein kinases and phosphatases that are responsible for signal regulation are difficult to study through biochemical and genetic methods. These traditional techniques fail to offer the acute spatial and temporal resolution that optical control provides. To that end, the objective of this research is to apply genetic code expansion to install light‐removable caging groups on critical residues for photo‐activation of phosphatase and kinase function. We hypothesized that protein activity can be restricted through the incorporation of a genetically encoded photocaged amino acid in place of 1) a conserved catalytic residue, 2) an active site‐adjacent residue, or 3) a protein docking site essential for enzyme/substrate interaction. The resulting precise temporal control, together with complete genetic specificity, will enable the dissection of intricate cell signaling mechanisms, including crosstalk regulation between signaling cascades. Here we describe the attempted photoregulation of SH2‐containing protein tyrosine phosphatase‐2 (SHP2) through three different sites of installation, using three unique unnatural amino acids. These approaches have been validated in conjunction with a fluorogenic phosphatase substrate and a live cell reporter. Additionally, we have demonstrated light activation of four unique isoforms of the mitogen‐activated protein kinase (MAPK), p38. We have utilized the isoform‐selective perturbation of kinase activity to reveal contradicting functions of p38 variants, resulting in either positive or negative crosstalk with the extracellular signal‐regulated kinase (ERK)/MAPK pathway by the γ/δ and α/β subsets, respectively. We conclude that genetic code expansion can be utilized to engineer photocontrol of enzymes via diverse methods, resulting in the precise examination of protein function within the context of complex cell signaling cascades, thereby delivering an additional understanding of kinase and phosphatase function.