Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.
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.
Ting
A Y
AY
Department of Pharmacology, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093, USA.
Kain
K H
KH
Klemke
R L
RL
Tsien
R Y
RY
D016475:3T3 Cells; D000818:Animals; D066246:ErbB Receptors / Q000235:genetics; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases