Review
Strategies for Engineering and Rewiring Kinase Regulation

https://doi.org/10.1016/j.tibs.2019.11.005Get rights and content

Highlights

  • Natural kinases are highly regulated by a diversity of mechanisms to ensure that activation occurs at the appropriate time and place.

  • Recent work has developed numerous strategies for engineering new kinase regulation, including controlling upstream signaling, manipulating kinase localization and association, sterically blocking the active site, and introducing novel allosteric control.

  • Light-based (optogenetic) regulation of kinases has been extended across the UV, visible, and infrared spectrum, allowing combinatorial regulatory inputs.

  • Structure-based design and sequence based coevolutionary analyses have emerged as productive strategies for identifying kinase surface sites with allosteric potential.

  • Kinase substrate specificity can be reprogrammed in cases where the recognition motif is well characterized.

Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.

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

References (81)

  • M. Hörner

    Optogenetic control of focal adhesion kinase signaling

    Cell Signal.

    (2018)
  • M. Kanwar

    Protein switch engineering by domain insertion

    Methods Enzymol.

    (2013)
  • S.M. Coyle

    Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation

    Cell

    (2013)
  • N. Halabi

    Protein sectors: evolutionary units of three-dimensional structure

    Cell

    (2009)
  • K.A. Reynolds

    Hotspots for allosteric regulation on protein surfaces

    Cell

    (2011)
  • S.M. Pearlman

    A mechanism for the evolution of phosphorylation sites

    Cell

    (2011)
  • A.C. Bishop

    Magic bullets for protein kinases

    Trends Cell Biol.

    (2001)
  • P. Creixell

    Unmasking determinants of specificity in the human kinome

    Cell

    (2015)
  • C. Chen

    Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity

    Mol. Cell

    (2014)
  • S. Regot

    High-sensitivity measurements of multiple kinase activities in live single cells

    Cell

    (2014)
  • G. Manning

    The protein kinase complement of the human genome

    Science

    (2002)
  • G.M. Rubin

    Comparative genomics of the eukaryotes

    Science

    (2000)
  • P. Cohen

    The origins of protein phosphorylation

    Nat. Cell Biol.

    (2002)
  • P.M. Fischer

    Approved and experimental small-molecule oncology kinase inhibitor drugs: a mid-2016 overview

    Med. Res. Rev.

    (2017)
  • S. Gross

    Targeting cancer with kinase inhibitors

    J. Clin. Invest.

    (2015)
  • P. Cohen

    Protein kinases – the major drug targets of the twenty-first century?

    Nat. Rev. Drug Discov.

    (2002)
  • J. Kuriyan et al.

    The origin of protein interactions and allostery in colocalization

    Nature

    (2007)
  • K. Oruganty et al.

    Design principles underpinning the regulatory diversity of protein kinases

    Philos. Trans. R Soc. Lond B Biol. Sci.

    (2012)
  • A. Ingles-Prieto

    Light-assisted small-molecule screening against protein kinases

    Nat. Chem. Biol.

    (2015)
  • X.X. Zhou

    Optical control of cell signaling by single-chain photoswitchable kinases

    Science (New York, N.Y.)

    (2017)
  • A.V. Leopold

    Optogenetically controlled protein kinases for regulation of cellular signaling

    Chem. Soc. Rev.

    (2018)
  • A.G. Goglia

    Optogenetic control of Ras/Erk signaling using the Phy–PIF system

    Methods Mol. Biol.

    (2017)
  • M.Z. Wilson

    Tracing information flow from Erk to target gene induction reveals mechanisms of dynamic and combinatorial control

    Mol. Cell

    (2017)
  • L.J. Bugaj

    Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras–Erk pathway

    Science

    (2018)
  • C.P. O'Banion

    Design and profiling of a subcellular targeted optogenetic cAMP-dependent protein kinase

    Cell Chem. Biol.

    (2018)
  • H. Wang

    LOVTRAP, an optogenetic system for photo-induced protein dissociation

    Nat. Methods

    (2016)
  • Y. Katsura

    An optogenetic system for interrogating the temporal dynamics of Akt

    Sci. Rep.

    (2015)
  • A.V. Leopold

    Neurotrophin receptor tyrosine kinases regulated with near-infrared light

    Nat. Commun.

    (2019)
  • S. Wend

    Optogenetic control of protein kinase activity in mammalian cells

    ACS Synth. Biol.

    (2014)
  • L. de Mena

    Bringing light to transcription: the optogenetics repertoire

    Front. Genet.

    (2018)
  • Cited by (0)

    View full text