Abstract
Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore, it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU, and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1-3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2 ± 5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and high-throughput inhibitor screening.
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