Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two proteinengineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1-and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.any physiological and pathological processes are regulated by protein kinases. Therefore, dissection of kinase functions helps to uncover molecular mechanisms and guides the development of new therapeutic strategies (1). However, progress in our understanding of kinase-mediated signaling is often hampered by the limitations of available tools (2). Pharmacological inhibitors of kinases have been valuable for the study of kinase functions, but they often do not provide the desired specificity and they are not available for the majority of kinases (1, 3). Also, the application of inhibitors is limited to the identification of processes affected by the inactivation of the kinase. Activation of a kinase is often achieved by the treatment of cells with growth factors or other extracellular stimuli. However, this approach triggers a multitude of parallel signaling pathways, which significantly complicates the analysis of individual kinase functions (4). Specific activation or inactivation of a kinase can be achieved by genetic modifications. However, this method is susceptible to compensatory artifacts and does not allow us to control the level and timing of kinase activation. Thus, development of novel tools that combine high specificity and tight temporal control of kinase activity remains a necessity. Under physiological conditions, kinases are activated for a finite period, and the duration of this activation is critical for eliciting specific biological outcomes (5, 6). Therefore, to mimic the biological activity of a kinase we need to use methods that allow for transient activation of a kinase with precise temporal control. An optogenetic approach has been successfully used for transient regulation of Raf kinase by dimerization (7). However, this method relies on the fact that Raf dimerization is required for its activation and thus has limited applicability to other kinases. Therefore, development of broadly applicable methods for tightly contro...

Highlights d SRC activation causes temporally distinct effects on the endothelial cell barrier d Initially, SRC causes endothelial barrier enhancement and VE cadherin rearrangement d VE cadherin phosphorylation on Y731 is required for SRCmediated barrier enhancement d Prolonged SRC activity cause barrier disruption

Protein tyrosine phosphatases (PTPases) are critical mediators of dynamic cell signaling. A tool capable of identifying transient signaling events downstream of PTPases is essential to understand phosphatase function on a physiological time scale. We report a broadly applicable protein engineering method for allosteric regulation of PTPases. This method enables dissection of transient events and reconstruction of individual signaling pathways. Implementation of this approach for Shp2 phosphatase revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2, mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediates cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. Broad application of this approach is demonstrated by regulating PTP1B and PTP-PEST phosphatases.