Trends in Biochemical Sciences
ReviewStrategies for Engineering and Rewiring Kinase Regulation
Section snippets
Engineered Kinase Regulation for Understanding Cell Biology, Pharmacology, and Evolution
EPKs play a central role in signal transduction by catalyzing the addition of the γ-phosphate of ATP to specific protein substrates at serine, threonine, or tyrosine residues. Phosphorylation in turn alters the localization pattern, activity, and/or interaction partners of the substrate. Consequently, this basic enzymatic reaction provides a fundamental mechanism for controlling cell differentiation, metabolism, cell-cycle progression, and motility, as well as the response to environmental cues
Strategies for Engineering Kinase Regulation
In recent years a diversity of strategies for engineering regulation have emerged which vary in their timescale of regulation, degree of reversibility, the input signal or stimulus, and their potential for perturbations to native kinase structure or function (Table 1, Key Table). Light-based, or optogenetic (see Glossary), kinases (optokinases) have become especially prevalent given the ease and speed of applying the stimulus [18,19]. We describe here four broad categories of engineered kinase
Structure-Based Design of Allosteric Regulation
In the structure-based design approach pioneered by Dagliyan and colleagues, allosteric insertion sites are selected to meet the following criteria: surface-exposed loops that form short, tight, connections between interacting structural units, and are relatively evolutionarily nonconserved [44]. Once candidate loops are identified, a regulatory domain is inserted. This can be the blue-light-controlled LOV2 domain or the ligand-inducible UniRapR system [32,44,45] (Figure 3A,B). Although
Evolution-Based Design of Allosteric Regulation
A different approach for identifying potential allosteric surfaces stems from statistical analysis of multiple sequence alignments (Figure 3E–H). Early work analyzing correlations in amino acid frequency across species found that coevolving networks of amino acids often link allosteric sites to active sites [49,50]. Using an approach called statistical coupling analysis (SCA) one can define sectors – collectively evolving groups of amino acids [51,52] (Figure 3E). In several protein families
Reprogramming Substrate Specificity
In addition to receiving novel inputs, kinases can be engineered to alter substrate specificity and thereby remodel signaling outputs (Figure 4). Altering substrate specificity has applications in synthetic rewiring of signaling pathways and can provide insight into evolutionary mechanisms and the consequences of disease-associated mutations. EPKs are broadly classified into two groups by the phosphoacceptor residue that they modify: the major group of kinases phosphorylates serine or threonine
Concluding Remarks and Future Perspectives
The above work presents a multifaceted toolkit for introducing synthetic kinase regulation. Although key questions remain (see Outstanding Questions), engineered kinases have rendered a large set of previously unfeasible cell biological experiments technically possible. For example, by coupling engineered kinases with recent advances in transcriptional and translational reporters, one can monitor information flow through the cell in real time [22]. Kinase translocation reporters (KTRs) are
Acknowledgments
We thank the numerous biochemists, geneticists, cell, structural, systems, synthetic, and computational biologists whose work on kinases laid the foundation for these recent developments. We regret that we could not discuss all of this work here owing to space constraints.
Glossary
- CRY2–Cib
- cryptochromes (CRYs) are a widely distributed class of photoreceptors that reduce a flavin adenine dinucleotide (FAD) chromophore in response to blue light. CRY2 of Arabidopsis thaliana exhibits light-dependent binding of the cryptochrome-interacting basic helix-loop-helix protein 1 (Cib).
- C-spine
- the catalytic spine; one of two hydrophobic connections between the N-lobe and C-lobe of the conserved protein kinase core, includes the adenine ring of bound ATP.
- DrBphP
- Deinococcus radiodurans
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