Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells - PubMed (original) (raw)
Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells
A Y Ting et al. Proc Natl Acad Sci U S A. 2001.
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.
Figures
Figure 1
General design of ratiometric indicators for monitoring tyrosine kinase activities (a). On phosphorylation by a kinase of interest, the phosphotyrosine binding domain (such as an SH2 domain) forms an intramolecular complex with the phosphotyrosine side chain, giving rise to a distance change between the two flanking GFPs that alters the FRET. Dephosphorylation reverses the FRET change. The domain structures of the individual indicators are shown in b. The three tyrosine kinases lie along the growth factor-stimulated signaling pathways depicted in c. In the scheme, indicators are boldfaced, the major tyrosine kinases are enlarged, and kinase inhibitors are italicized. When the purified EGFR indicator was treated with EGFR kinase and ATP in vitro, a 25% increase in emission ratio was observed (d). Reversal of the FRET change was observed after removal of the kinase and addition of YOP tyrosine phosphatase (data not shown for the Src and Abl indicators). Site-directed mutagenesis was used to examine the mechanistic basis of the FRET response of each of the indicators. The percentage emission ratio change for each indicator and mutant in response to in vitro phosphorylation is shown in_e_. Indicator specificity was tested by using a panel of different kinases as shown in e.
Figure 2
B82 cells transfected with the EGFR indicator responded to EGF stimulation with an ≈30% increase in yellow to cyan emission ratio (transition from blue to red pseudocolor; a). The FRET change visibly propagated from the plasma membrane to the rest of the cytosol in ≈3 min. The far left shows the fluorescence from the YFP channel only. Three different subcellular regions are circled in red and correspond to the indicated lines in the time course graph shown in_b_. The red x's on the graph denote the times at which each subcellular region reached 50% of its maximal FRET change. Antiphosphotyrosine Western analysis of immunoprecipitated EGFR reporter (using α-GFP) showed an increase in phosphorylation level on EGF treatment (c). Lane 1, unstimulated cells; lane 2, cells treated with EGF for 10 min; lane 3, EGF treatment, followed by washout and addition of AG1478 for 20 min. The EGFR indicator was a reversible reporter (d). On removal of EGF, the FRET change slowly reversed to its original level in ≈30 min. Reversal was accelerated by adding the EGFR inhibitor AG1478. The FRET response of the Src reporter in HeLa cells stimulated with EGF is shown in_e_. The far left shows the YFP-only image. This indicator, too, was reversible, giving a recovery in FRET change after removal of extracellular EGF and addition of AG1478 (f).
Figure 3
Cellular response of the Crk-based reporter. Wild-type MEFs stimulated with H2O2 displayed a FRET increase in the cytosol over 20 min but no change in the nucleus (a). Null cells did not display such an increase. An antiphospho-Y221Crk Western blot of crude cell lysates from NIH 3T3 cells transfected with the Crk-based indicator shows an increase in phosphorylation of the indicator on PDGF stimulation (b). Phosphorylation is suppressed by pretreating cells with the Abl inhibitor STI-571. The emission ratio time course (c) for different regions of the PDGF-stimulated cell depicted in d shows a cytoplasmic increase in FRET followed by a dramatic FRET increase within the PDGF-induced membrane ruffles. Corresponding images of the YFP fluorescence show that the Abl reporter concentrates in the membrane ruffles (e).
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