Generation of a Light Inhibited Src Kinase through Insertion of LOV into the Catalytic Domain

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Src is a non-receptor tyrosine kinase that participates in a diverse spectrum of signaling pathways. Because the interactions of Src with activators and downstream ligands depend on the spatio-temporal dynamics of Src activation, it will be valuable to control the timing and location of activation in vivo with light. Through insertion of the light oxygen voltage (LOV) domain into a conserved portion of the kinase catalytic domain, we have generated a genetically encoded analog of Src that is reversibly inhibited upon irradiation at wavelengths between 400 and 500 nm. With optimized linkers, the insertion minimally perturbed Src catalytic activity in the dark, but led to a greater than 2-fold reduction in activity upon irradiation. Molecular dynamics studies indicate that the light-induced unwinding of the LOV Jα-helix results in narrowing of the ATP binding site. The activation loop and beta-sheet move closer to each other, emulating the structure of the inactive state. Photoinhibition of Src reduced fibroblast migration rates and the frequency of protrusion. Cells transformed by overexpression of LOV-Src in the dark could be reverted to a normal phenotype upon irradiation. Structural studies and insertion of other engineered folds into the same Src site indicate that this strategy can be broadly applied to other kinases.