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Allosteric Regulation of Protein Kinases Using Optogenetics

Activation of single protein kinase can result in a very distinct signaling output depending on the duration, location, and frequency of its activation in the cell. However, interrogation of kinase‐mediated processes remains challenging due to the paucity of tools that precisely regulate kinases in space and time. To overcome existing limitations, we developed a novel approach for light‐mediated regulation of kinases. We engineered a light‐regulated allosteric switch (LightR), a small domain based on Vivid (VVD) photoreceptor, that undergoes conformational changes upon illumination with blue light. Insertion of LightR at a specific location in the catalytic domain of Src kinase enables light‐mediated control of its activity. In the dark, engineered LightR‐Src is inactive. Illumination of cells with blue light leads to robust activation of the kinase. Activation of LightR‐Src is specific, fast, and reversible. This allows us to control duration of kinase activation as well as induce repeated cycles of kinase activation/inactivation in living cells. Moreover, the level of activation can be controlled by changing light intensity. Specific modifications of the LightR domain allow us to modulate the activation/inactivation kinetics and the level of activity of LightR‐Src, enabling additional “tuning” of the engineered kinase and its optimization for different applications. Activation of LightR‐Src in living cells induces robust cell spreading and protrusive activity, which are known Src‐induced phenotypes described in previous studies. Importantly, the morphological changes stop as soon as the kinase is inactivated, demonstrating reversibility of the manipulated signaling. Repeated activation/inactivation of LightR‐Src induces cycles of morphodynamic activity of the cells, showing the capability of this tool to mimic oscillation of kinase signaling in living cells. Localized illumination of cells induces local formation of protrusions and cell polarization towards the light, demonstrating spatial regulation of LightR‐Src. Our study also reveals that continuous local activation of LightR‐Src at the cell periphery induced local oscillations of protrusive activity with defined periodicity. Insertion of LightR domain at a homologous position in Abl kinase also enabled light‐mediated regulation of its activity. These results, and the high structural similarity among catalytic domains of kinases, suggest broad applicability of this tool for regulation of different kinases. Furthermore, this approach could be potentially employed to control the activity of other enzymes in time and space.