Genetically Encoded Fluorescent Reporters of Protein Tyrosine Kinase Activities in Living Cells

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2001 Dec 26

PMID 11752449

Citations 138

Authors

A Y Ting

K H Kain

R L Klemke

R Y Tsien

Affiliations

Soon will be listed here.

Abstract

The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.

Citing Articles

Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals.

Gest A, Sahan A, Zhong Y, Lin W, Mehta S, Zhang J Chem Rev. 2024; 124(22):12573-12660.

PMID: 39535501 PMC: 11613326. DOI: 10.1021/acs.chemrev.4c00293.

Perspectives on Synthetic Protein Circuits in Mammalian Cells.

Aldrete C, An C, Call C, Gao X, Vlahos A Curr Opin Biomed Eng. 2024; 32.

PMID: 39372446 PMC: 11448451. DOI: 10.1016/j.cobme.2024.100555.

On-demand expansion fluorescence and photoacoustic microscopy (ExFLPAM).

Mu X, Ma C, Mei X, Liao J, Bojar R, Kuang S Photoacoustics. 2024; 38:100610.

PMID: 38726025 PMC: 11079527. DOI: 10.1016/j.pacs.2024.100610.

Investigating Heterogeneous Cell-Cycle Progression Using Single-Cell Imaging Approaches.

Yang H Methods Mol Biol. 2024; 2740:263-273.

PMID: 38393481 DOI: 10.1007/978-1-0716-3557-5_16.

Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.

Sanchez C, Ramirez A, Hodgson L J Microsc. 2024; .

PMID: 38357769 PMC: 11324865. DOI: 10.1111/jmi.13270.

References

Plattner R, Kadlec L, DeMali K, Kazlauskas A, Pendergast A . c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF. Genes Dev. 1999; 13(18):2400-11. PMC: 317022. DOI: 10.1101/gad.13.18.2400. View

Ng T, Squire A, Hansra G, Bornancin F, Prevostel C, Hanby A . Imaging protein kinase Calpha activation in cells. Science. 1999; 283(5410):2085-9. DOI: 10.1126/science.283.5410.2085. View

Cotton G, Muir T . Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation. Chem Biol. 2000; 7(4):253-61. DOI: 10.1016/s1074-5521(00)00100-9. View

Sun X, Majumder P, Shioya H, Wu F, Kumar S, Weichselbaum R . Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species. J Biol Chem. 2000; 275(23):17237-40. DOI: 10.1074/jbc.C000099200. View

Escalante M, Courtney J, Chin W, Teng K, Kim J, Fajardo J . Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis. J Biol Chem. 2000; 275(32):24787-97. DOI: 10.1074/jbc.M000711200. View

Miyawaki A, Tsien R . Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 2000; 327:472-500. DOI: 10.1016/s0076-6879(00)27297-2. View

Verveer P, Wouters F, Reynolds A, Bastiaens P . Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane. Science. 2000; 290(5496):1567-70. DOI: 10.1126/science.290.5496.1567. View

Yaffe M, Elia A . Phosphoserine/threonine-binding domains. Curr Opin Cell Biol. 2001; 13(2):131-8. DOI: 10.1016/s0955-0674(00)00189-7. View

Kain K, Klemke R . Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk-CAS complexes. J Biol Chem. 2001; 276(19):16185-92. DOI: 10.1074/jbc.M100095200. View

10.

Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A . Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature. 2001; 411(6841):1065-8. DOI: 10.1038/35082594. View

11.

Griesbeck O, Baird G, Campbell R, Zacharias D, Tsien R . Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001; 276(31):29188-94. DOI: 10.1074/jbc.M102815200. View

12.

Kurokawa K, Mochizuki N, Ohba Y, Mizuno H, Miyawaki A, Matsuda M . A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo. J Biol Chem. 2001; 276(33):31305-10. DOI: 10.1074/jbc.M104341200. View

13.

Zhang J, Ma Y, Taylor S, Tsien R . Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A. 2001; 98(26):14997-5002. PMC: 64972. DOI: 10.1073/pnas.211566798. View

14.

Chen W, LAZAR C, Poenie M, Tsien R, Gill G, Rosenfeld M . Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. Nature. 1987; 328(6133):820-3. DOI: 10.1038/328820a0. View

15.

Czernik A, Girault J, Nairn A, Chen J, Snyder G, Kebabian J . Production of phosphorylation state-specific antibodies. Methods Enzymol. 1991; 201:264-83. DOI: 10.1016/0076-6879(91)01025-w. View

16.

Ridley A, Hall A . The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992; 70(3):389-99. DOI: 10.1016/0092-8674(92)90163-7. View

17.

Feller S, Knudsen B, Hanafusa H . c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 1994; 13(10):2341-51. PMC: 395099. DOI: 10.1002/j.1460-2075.1994.tb06518.x. View

18.

Opresko L, Chang C, Will B, Burke P, Gill G, Wiley H . Endocytosis and lysosomal targeting of epidermal growth factor receptors are mediated by distinct sequences independent of the tyrosine kinase domain. J Biol Chem. 1995; 270(9):4325-33. DOI: 10.1074/jbc.270.9.4325. View

19.

Mayer B, Hirai H, Sakai R . Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases. Curr Biol. 1995; 5(3):296-305. DOI: 10.1016/s0960-9822(95)00060-1. View

20.

Romoser V, Hinkle P, Persechini A . Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem. 1997; 272(20):13270-4. DOI: 10.1074/jbc.272.20.13270. View