@article{0a8ff3d5f4f342f99db02dc7fde54799,
title = "Optimizing photoswitchable MEK",
abstract = "Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.",
keywords = "ERK signaling, Optogenetics, Photoswitchable MEK",
author = "Patel, {Aleena L.} and Eyan Yeung and McGuire, {Sarah E.} and Wu, {Andrew Y.} and Toettcher, {Jared E.} and Burdine, {Rebecca D.} and Shvartsman, {Stanislav Y.}",
note = "Funding Information: ACKNOWLEDGMENTS. We thank Phillip Johnson and the Laboratory Animal Resources (LAR) staff for zebrafish care; Michael Lin for plasmids; Dr. Gary Laevsky and the Molecular Biology Confocal Microscopy Facility, which is a Nikon Center for Excellence, for microscopy support; and the Alexei Korrennykh laboratory (Molecular Biology, Princeton University) for the use of their qPCR machine and assistance with the thermo shift experiment. We thank Granton Jindal and all members of the S.Y.S. and R.D.B. laboratories for helpful discussions. A.L.P. thanks Rajeshwari Enjeti for her contributions to developing p-Smad5 immunofluorescence and quantification procedures in zebrafish. This work was supported by the National Science Foundation Graduate Research Fellowship Program Grant DGE-1656466 (to A.L.P.). S.Y.S. and R.D.B. were supported by NIH Grant GM086537. J.E.T. was supported by the National Science Foundation CAREER Award 1750663. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Funding Information: We thank Phillip Johnson and the Laboratory Animal Resources (LAR) staff for zebrafish care; Michael Lin for plasmids; Dr. Gary Laevsky and the Molecular Biology Confocal Microscopy Facility, which is a Nikon Center for Excellence, for microscopy support; and the Alexei Korrennykh laboratory (Molecular Biology, Princeton University) for the use of their qPCR machine and assistance with the thermo shift experiment. We thank Granton Jindal and all members of the S.Y.S. and R.D.B. laboratories for helpful discussions. A.L.P. thanks Rajeshwari Enjeti for her contributions to developing p-Smad5 immunofluorescence and quantification procedures in zebrafish. This work was supported by the National Science Foundation Graduate Research Fellowship Program Grant DGE-1656466 (to A.L.P.). S.Y.S. and R.D.B. were supported by NIH Grant GM086537. J.E.T. was supported by the National Science Foundation CAREER Award 1750663. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Publisher Copyright: {\textcopyright} 2019 National Academy of Sciences. All rights reserved.",
year = "2019",
month = dec,
day = "17",
doi = "10.1073/pnas.1912320116",
language = "English (US)",
volume = "116",
pages = "25756--25763",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "51",
}