Manipulation of Endogenous Kinase Activity in Living Cells Using Photoswitchable Inhibitory Peptides

Abstract

Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.

Small Molecule Gated Split-Tyrosine Phosphatases and Orthogonal Split-Tyrosine Kinases

“…68−72 In this vein, Hahn and co-workers have recently reported a LOV2 domain based design of light activated kinase inhibitors. 72 The small molecule activated orthogonal control over specific enzyme catalyzed phosphorylation and dephosphorylation events described herein complements ongoing efforts toward understanding and controlling the phosphoproteome using a variety of traditional and nontraditional approaches. 18,19,73−82 More generally, the sequence dissimilarity based approach for identification of fragmentation sites to control split-protein function may also prove to be applicable to a variety of enzyme families involved in post-translational modifications such as acetylases and deacetylases; 83 methyltransferases and demethylases; 84,85 and ubiquitin ligases and deubiquitinases.…”

Synthetic protein switches: design principles and applications

“…Proof-ofconcept was first realized with the small GTPase Rac1, for which light-dependent conformational changes controlled access of Rac1 to its effector protein kinase PAK and, thus, cell motility [59] (Figure 4A). Similar types of light-inducible protein switch have since been constructed to activate kinase inhibitors [60], control membrane and promoter localization through PDZ affinity-clamp interactions [61,62], control ion channel permeability [63], regulate gene expression and proteosomal degradation in E. coli through ipaA-vinculin and SsrA-SspB interactions [64] and control nuclear localization [65]. In the latter two cases, Ja of AsLov2 was engineered such that the molecular specificity feature-binding of the AsLov2 core overlapped with effector binding.…”

Optogenetic approaches to cell migration and beyond

“…Peptides have been appended to the end of the helix where light-induced unwinding of the helix freed them to interact with targets. Fragments of formin autoinhibitory domains that activate endogenous mDia1 [17, 18], an inhibitor of protein kinase A, and a myosin light chain kinase inhibitor [19] have been caged using this approach. Molecular modeling was used to incorporate residues from the vinculin-binding peptide IPA at several different places in the Ja helix, at positions where they did not interfere with helix-LOV binding, but were capable of interacting with vinculin once the helix unwound [20].…”