Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 24;355(6327):836-842.
doi: 10.1126/science.aah3605.

Optical control of cell signaling by single-chain photoswitchable kinases

Affiliations

Optical control of cell signaling by single-chain photoswitchable kinases

Xin X Zhou et al. Science. .

Abstract

Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. A modular and generalizable design for photoswitchable kinases
(A) Photodissociable dimeric Dronpa (pdDronpa) variants were engineered from tetrameric DronpaN145. Residues 145 and 158 were further mutated to tune affinity. (B) Structural model of ps(ΔNT)MEK1 in the pre-illuminated state, showing the MEK1 core kinase domain with active site (asterisk) caged by pdDronpa1 domains attached at the NT and the GH loop (rendering based on PDB files 1S9J for MEK1 and 2Z6Y for Dronpa). Note ps(ΔNT)MEK1 contains constitutively activating mutations as well. (C) Light-dependent induction of ERK phosphorylation (pERK) by psMEK1 and psMEK1tight. (D) Structural alignment of MEK1 (PDB 1S9J) with MEK2 (PDB 1S9I). (E) Light-dependent induction of pERK by psMEK2. (F) Structural alignment of MEK1 (PDB 1S9J) with Raf1 (PDB 3MOV). (G) Light-dependent induction of pERK by psRaf1. Note psRaf1 contains a C-terminal CAAX motif for constitutive membrane localization. In (C,E,G), cells were illuminated by 20-mW/cm2 cyan light for 2 min. Protein was detected via an N-terminal HA tag, and lysate loading was monitored by blotting for GAPDH. Serum stimulations were for 5–10 min. Error bars represent standard error of the mean (s.e.m.), n = 3. (H) psMEK1 activation can be temporally and reversibly controlled. Upper panels, intrinsic pdDronpa fluorescence in psMEK1. Lower panels, mRuby2 fluorescence of the ERK KTR sensor. Cells were illuminated with 200-mW/cm2 cyan light for 1 min after the 0- and 60-min timepoints, and with 200-mW/cm2 violet light for 3 s after the 30-min timepoint. pdDronpa fluorescence was imaged immediately after each light stimulation. Scale bar, 20 μm. Chart, quantification of cytosolic/nuclear KTR fluorescence over time. Error bars represent s.e.m. of imaged cells.
Fig. 2
Fig. 2. Cells expressing psMEK1 and ERK KTR sensor enables all-optical cell-based assay for MEK and ERK inhibitors
(A) In native pathway, chemical ligands such as EGF activates receptor kinases EGFR, which then activates Ras. Activated Ras binds and activates Raf-1, which leads to MEK activation. In the synthetic pathway, psMEK1 is solely controlled by light and no longer respond to upstream activations. (B) Proposed all-optical cell-based assay for MEK and ERK inhibitors. Cells expressing psMEK1-P2A-ERK KTR-mRuby would be first incubated with drug candidates and then stimulated with light. The distribution of ERK KTR-mRuby2 would be monitored to determine whether light could induce ERK phosphorylation in the presence of the drug. (C) The assay allowed determination of inhibitors specifically targeting MEK and ERK. Upper panels, 1 min of cyan light stimulation at 200-mW/cm2 switched off pdDronpa fluorescence; lower panels, ERK KTR-mRuby2 translocation was monitored. We observed that cells incubated with ERK inhibitor SCH772984 and VX-11e, and MEK inhibitor U0126, did not show ERK KTR-mRuby2 translocation in response to psMEK1 activation, but cells incubated with DMSO or inhibitors against upstream MEK activators Raf1 or EGFR, Sorafenib and Lapatinib, showed ERK KTR. Scale bar, 20 μm.
Fig. 3
Fig. 3. Photoswitchable kinases allows fine dissection of Raf-MEK signaling and identification of a novel fast feedback mechanism
(A) 2 min illumination by 20-mW/cm2 500-nm light effectively switched off pdDronpa fluorescence. 3 s of illumination by 10-mW/cm2 400-nm light fully recovered pdDronpa fluorescence. Scale bar, 20 μm. (B) Cells stably expressing psRaf1 were illuminated by 500-nm light for 1–2 minutes followed by illumination with 400-nm light. Cell are then collected at 5,10,15 min and immunoblotted. (C) MEK phosphorylation is downregulated over time, and downregulation requires PP2A/PP1. Lysis time means the time from the start of the stimulation to cell lysis. (D) MEK phosphorylation is downregulated over time, and downregulation requires ERK activity. Lanes 1–4, decreases of pMEK-218/222 occurred over time after transient activation of psRaf1 (by 1–2 min of 500-nm light followed by 3 s 400-nm light), demonstrating that dephosphorylation of pMEK-218/222 occurs. Right, downregulation of pMEK did not occur in cells incubated with ERK inhibitor SCH772984, indicating that ERK regulates the phosphatase activity. (E) Quantification of pMEK-218/222 over time in cells treated with DMSO, okadaic acid, or SCH772984. Error bars are standard error of the mean (n = 3). Unpaired two-sided t-tests were used for statistical analysis. (F) Simplified diagram of known positive (blue arrows) and negative (gray arrows) regulatory pathways mediating signaling from growth factor receptors to ERK. The new proposed pathway of ERK-induced dephosphorylation of pMEK218/222 is marked with red arrows. Dashed arrows indicate protein transitions, while solid arrows indicate regulatory effects.
Fig. 4
Fig. 4. Optical control of kinase activity in living animals
(A) Wild-type C. elegans worms showed no swollen tails even when grown in 500-nm light, while 80% of worms expressing DronpaK145-active(ΔNT)MEK1 and DronpaK145-Raf1-CAAX exhibited swollen tails in the dark (n = 146). 100% of worms expressing psMEK1 in the dark had normal tail shape (n = 50), while 73% of worms expressing psMEK1 in cyan light showed tail swelling (n = 15). Among worms expressing psRaf1, 98% had normal tail shape in the dark (n = 83), while 79% showed tail swelling in light (n = 34). (B) In wild-type worms, synaptic vesicles marked by RAB-3-GFP localize exclusively in the axon. In cyy-1 mutants, RAB-3-GFP is partly mislocalized to the dendrite. In cyy-1;cdk-5 double-mutant worms, nearly all RAB-3-GFP localized to the dendrite. Green and red arrows mark vesicles located in the axon and dendrite, respectively. (C) Light-dependent restoration of CDK5 function in cdk-5;cyy-1 worms. Nearly all RAB-3-mCherry in cyy-1;cdk-5 worms expressing psCDK-5 localized in the dendrite in the absence of light (n = 11), similar to cyy-1;cdk-5 worms. Cyan illumination restored axonal localization of some vesicles (n = 10), similar to the phenotype of cyy-1 worms. The light effect was statistically significant (p = 0.006, unpaired two-sided t-test). Error bars represent standard error of the mean. Worms were exposed to 400-nm light at 0.7-mW/cm2 for 24–48 h before imaging.

Similar articles

Cited by

References

    1. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002;298:1912–1934. - PubMed
    1. Bergeron JJ, Di Guglielmo GM, Dahan S, Dominguez M, Posner BI. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction. Annu Rev Biochem. 2016 - PubMed
    1. Purvis JE, Lahav G. Encoding and decoding cellular information through signaling dynamics. Cell. 2013;152:945–956. - PMC - PubMed
    1. Shimizu-Sato S, Huq E, Tepperman JM, Quail PH. A light-switchable gene promoter system. Nat Biotechnol. 2002;20:1041–1044. - PubMed
    1. Levskaya A, Weiner OD, Lim WA, Voigt CA. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature. 2009;461:997–1001. - PMC - PubMed

Publication types

MeSH terms

Substances