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Mimicking transient activation of protein kinases in living cells

Significance Nearly all eukaryotic physiological events are regulated by protein kinases. Aberrations in functions of kinases are linked to numerous diseases. Significant progress has been made in dissecting the role of many kinases. However, understanding how specific kinases orchestrate complex signaling events remains challenging. Here we present a method to control the timing and the duration of kinase activity. This system provides the ability to mimic physiologically relevant transient activation of a kinase. We demonstrate the specificity and efficiency of this method by regulating tyrosine kinase (c-Src) and serine/threonine kinase (p38α). By using this approach, we identify morphological changes induced by transient activation of Src and demonstrate the role of sequential Src-PI3K and Src-Rac1 signaling in regulation of cell morphology. 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 protein-engineering 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.