A Light-Activated Probe of Intracellular Protein Kinase Activity

Abstract

The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points.

Photoactivatable Fluorophores

“…This general design logic is extremely versatile and can easily be adapted to most of the main families of fluorescent probes with the aid of chemical synthesis. Indeed, photoactivatable acridinone [46], anthracene [47], benzofurazan [48], borondipyrromethene [49], chromone [50], coumarin [51][52][53][54][55][56], dihydrofuran [57][58][59], fluorescein [60][61][62][63][64][65][66][67], naphthalene [68], pyranine [69], pyrazolines [70], resorufin [71], rhodamine [66,[72][73][74][75][76], thioxanthone [77] and xanthone [78] derivatives have been developed on the basis of these operating principles. Furthermore, this general strategy has been adapted to activate also the luminescence of conjugated oligomers [79] and semiconductor quantum dots [80,81].…”

“…As a result, these functional fluorescent probes permit the implementation of imaging and spectroscopic protocols that are otherwise inaccessible with conventional fluorophores. Indeed, photoactivatable fluorophores are routinely used as calibration standards in photolysis experiments [131][132][133], to probe the activity and dynamics of biomolecular systems [48,49,55,61,62,68,71,[134][135][136][137][138][139][140][141][142], to monitor the flow dynamics of fluids [143][144][145][146][147][148][149][150][151][152], and to reconstruct subdiffraction images [57-59, 74-76, 98, 99, 103, 104, 129, 153-160]. The common theme of all these strategies is the interplay of activating and exciting beams to switch fluorescence on within a defined region of space at a given interval of time.…”

Photolabile amphiphiles with fluorogenic thioxanthone-dithiane functionality: synthesis and photoinduced fragmentation in micelles

“…The mixture was stirred at 20°C overnight, after that the solvent was evaporated, and the residue was passed through alumina (CH 2 Cl 2 /THF 10: 1) to furnish the product as a yellow oil, 0.29 g (53%). 1 4 (10 g, 0.26 mol) was suspended in 500 mL of THF. Diethyl 2,2-bis(hexadecyl)malonate 15 (70 g, 0.115 mol), obtained by alkylation of diethyl malonate via previously described procedures, was dissolved in 100 mL of THF and added to the suspension at 20°C.…”

“…Besides the fact that this solution is a somewhat artificial hybrid of old technologies, the caveat is that the UV-Vis absorption of the caged fluorophore is often higher than the photoremovable group, which inevitably reduces the quantum efficiency of release and may cause other complications. Nevertheless, compact fluorogenic caging groups show great promise as demonstrated by numerous accounts, including an elegant solution by Lawrence [4] for probing intracellular protein kinase activity with a light activated probe. The probe has the enzyme substrate, outfitted with a fluorophore and, in the immediate vicinity of this fluorophore -a strategically placed serine residue, decorated with the nitroveratryl.…”

A multifunctional β-cyclodextrin-conjugate photodelivering nitric oxide with fluorescence reporting

“…This elegant strategy relies on the simultaneous photorelease of the desired bioactive species, i.e. NO, and a fluorescent component (the reporter) from the same nonfluorescent precursor (Veldhuyzen et al, 2003;Pellois et al, 2004;Weinstain et al, 2010). The release process can be thus easily quantified by monitoring the fluorescence emission of the reporter, which acts exactly as an optical counter of the bioactive species.…”