Photoactivatable Reporter to Perform Multiplexed and Temporally Controlled Measurements of Kinase and Protease...

Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.