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Review
. 2019 Oct 4;294(40):14814-14822.
doi: 10.1074/jbc.REV119.006177. Epub 2019 Aug 21.

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer

Wei Lin  1 , Sohum Mehta  1 , Jin Zhang  2
Affiliations
Review

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer

Wei Lin et al. J Biol Chem. .

Abstract

Protein kinase signaling networks stringently regulate cellular processes, such as proliferation, motility, and cell survival. These networks are also central to the evolution and progression of cancer. Accordingly, genetically encoded fluorescent biosensors capable of directly illuminating the spatiotemporal dynamics of kinase signaling in live cells are being increasingly used to investigate kinase signaling in cancer cells and tumor tissue sections. These biosensors enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Herein, we first review common design strategies for kinase activity biosensors, including signaling targets, biosensor components, and fluorescent proteins involved. Subsequently, we discuss applications of biosensors to study the biology and management of cancer. These versatile molecular tools have been deployed to study oncogenic kinase signaling in living cells and image kinase activities in tumors or to decipher the mechanisms of anticancer drugs. We anticipate that the diversity and precision of genetically encoded biosensors will expand their use to further unravel the dysregulation of kinase signaling in cancer and the modes of actions of cancer-targeting drugs.

Keywords: biosensor; cancer; cell signaling; fluorescence resonance energy transfer (FRET); fluorescent protein; in vivo imaging; kinase signaling; phosphorylation; posttranslational modification.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Genetically encoded biosensors for visualizing kinase activity in living cells. A, FRET-based biosensors utilize a kinase-inducible molecular switch to modulate the relative distance and orientation of a pair of FPs in response to changes in biosensor phosphorylation, thereby coupling endogenous kinase activity to biosensor FRET efficiency. As shown here, the molecular switch is often constructed by fusing a substrate peptide for a specific kinase to a PAABD, such that recognition of the phosphorylated substrate by the PAABD drives a conformational change in the biosensor. B, single-fluorophore kinase biosensors have recently been generated in which the substrate peptide and PAABD are inserted within the sequence of a cpFP, whereby the recognition of the phosphorylated substrate by the PAABD distorts the barrel of the cpFP, thus altering its photophysical behavior. In the example shown here, phosphorylation of the sensor leads to a shift in the maximum excitation wavelength of cpGFP from 400 to 480 nm, producing an excitation-ratiometric response. C, an alternative strategy for constructing single-fluorophore biosensors retains the same overall configuration of FRET-based sensors but replaces the FRET FP pair with a pair of dimerization-dependent FPs. This design also relies on the molecular switch to modulate the proximity of the dimerization-dependent FPs, as bringing FP-A and B into close proximity results in enhanced fluorescence emission by FP-A and has been used to develop red-fluorescent kinase sensors. D, instead of relying on a conformational change, translocation-based kinase biosensors use phosphorylation to control the behavior of nuclear localization and export signals that are incorporated within the substrate sequence. At rest, the dephosphorylated biosensor accumulates in the nucleus, whereas elevations in kinase activity drive the translocation of the biosensor into the cytosol, whereby the ratio of nuclear-to-cytoplasmic fluorescence serves as a dynamic readout of kinase activity.
Figure 2.
Figure 2.
Using genetically encoded kinase biosensors to study cancer. A, studying cancer-associated kinases at the single-cell level. PKC has long been considered to be an oncogene due to the ability of phorbol esters, which potently activate PKC, to promote tumorigenesis. However, when Antal et al. (44) probed the role of PKC in cancer by examining the effect of various cancer-associated mutations on PKC activity using the FRET-based PKC activity reporter CKAR, most mutations were found to either have no effect on PKC activity or be loss-of-function mutations. For example, among 12 PKCα mutations tested, seven exhibited reduced activity compared with WT PKCα, whereas the remaining five showed no difference; no gain-of-function mutants were observed. Their results strongly suggest that PKC is in fact a tumor suppressor and highlight the importance of using biosensors to dissect cancer-associated signaling in the native context of living cells. B, illuminating heterogenous signaling in tumor tissues. Tumors are highly complex and intricately organized tissues. Interactions among cells within different parts of the tumor can form diverse microenvironments associated with unique signaling dynamics, and genetically encoded biosensors can be powerful tools for elucidating the mechanisms of this heterogeneity. For example, by using the FRET-based EGFR biosensor Picchu-X to visualize EGFR signaling in tumor xenografts, Weitsman et al. (27) were able to observe substantial heterogeneity in EGFR activation within the tumor. Because they were imaging biosensor responses in intact tissue, they were further able to correlate the responses to the proximity of different tumor structures and cell types, revealing that proximity to infiltrating macrophages was strongly associated with elevated EGFR activity in tumor cells. C, biosensors enable high-content screening for pathway regulators. Because they can directly and sensitively report on endogenous kinase activities, genetically encoded biosensors are increasingly being used in high-content studies to dissect signaling pathways and identify drug targets. Azad et al. (43) recently applied this approach using their newly developed bioluminescent LATS1 sensor to identify upstream regulators of the Hippo pathway. By culturing biosensor-transfected HEK293 cells in microtiter plates and treating them with a large panel of kinase inhibitors, they were able to identify both activators and inhibitors of LATS1 kinase, thus providing new details of Hippo pathway regulation.
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References

    1. Wilson L. J., Linley A., Hammond D. E., Hood F. E., Coulson J. M., MacEwan D. J., Ross S. J., Slupsky J. R., Smith P. D., Eyers P. A., and Prior I. A. (2018) New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer Res. 78, 15–29 10.1158/0008-5472.CAN-17-2291 - DOI - PubMed
    1. Hunter T. (2009) Tyrosine phosphorylation: thirty years and counting. Curr. Opin. Cell Biol. 21, 140–146 10.1016/j.ceb.2009.01.028 - DOI - PMC - PubMed
    1. Hornbeck P. V., Kornhauser J. M., Latham V., Murray B., Nandhikonda V., Nord A., Skrzypek E., Wheeler T., Zhang B., and Gnad F. (2019) 15 years of PhosphoSitePlus® integrating post-translationally modified sites, disease variants and isoforms. Nucleic Acids Res. 47, D433–D441 10.1093/nar/gky1159 - DOI - PMC - PubMed
    1. Du Z., and Lovly C. M. (2018) Mechanisms of receptor tyrosine kinase activation in cancer. Mol. Cancer 17, 58 10.1186/s12943-018-0782-4 - DOI - PMC - PubMed
    1. Roskoski R., Jr. (2019) Targeting ERK1/2 protein-serine/threonine kinases in human cancers. Pharmacol. Res. 142, 151–168 10.1016/j.phrs.2019.01.039 - DOI - PubMed

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