Protein tyrosine kinases (PTKs) are well-established participants in signaling pathways that drive and maintain a host of normal and aberrant cellular phenotypes. The complexities associated with the regulation of PTK activation are most appropriately examined in the intracellular environment, conditions that allow PTK activity to be correlated with cellular behavior. Consequently, there has been intense interest in developing reporters that can fluorescently sense PTK activity in real time and under conditions that are consistent with living cells.[1,2] Recently, we described a strategy for the design of PTK reporters that relies upon the phosphorylation-induced disruption of noncovalent interactions between the kinasetargeted Tyr residue and a proximal pyrene moiety.[3] However, the latter, like all other PTK sensors described to date, is immediately susceptible to phosphorylation. The inability to control sensing activity precludes an assessment of intracellular kinase action as a function of the cell cycle in particular, and environmentally-stimulated behaviors in general. We report the first example of a light-regulated protein tyrosine kinase sensor. [6] are relatively bright fluorophores with high quantum yields, good resistance to photobleaching and distinct spectral properties. The cascade yellow Y−2 derivative 3 exhibits a 2.7-fold enhancement in fluorescence intensity upon phosphorylation. By contrast, the corresponding cascade yellow-substituted derivative at Y+3 (i.e. 4) fails to furnish a fluorescence response. In addition, Oregon green (5) and cascade blue (6) display 2-fold enhancements at Y−2 but more modest changes at Y+3. NOESY experiments on 3 and its phosphorylated counterpart revealed that the Tyr residue in both peptides exhibits through-space interactions with the fluorophore (Supporting Information), with significantly stronger interactions in the nonphosphorylated species. Indeed, chemical shift data demonstrates that the free Tyr phenol in 3 is significantly more shielded relative to Tyr residues in general [7,8] and the phosphoTyr moiety in phospho-3 in particular. These results suggest that the ring current of the proximal fluorophore electronically dlawrenc@aecom.yu.edu. Supporting Information AvailableExperimental details of peptide synthesis and characterization, and enzyme and cell-based assays. The nature of the fluorophore influences the efficiency of phosphorylation (Supporting Information). The negatively charged cascade blue derivative 6 displays a low K m value (3.0 ± 0.2 μM), which may be a good barometer of enzyme affinity since the Src kinase exhibits a proclivity for negatively charged substrates. By contrast, the partially positively charged cascade yellow derivative 3 exhibits a significantly higher K m value (300 ± 10 μM). The spectral characteristics of these fluorophores should prove useful for sampling the activity of more than one protein kinase or for use in conjunction with other probes. For example, 5 (λ ex = 495 nm; λ em = 520 nm) and 6 (λ ex = 400 nm; λ em ...