Protein-Phosphorylation-Responsive Polymeric Nanoparticles for Imaging Protein Kinase Activities in Single Living Cells

摘要

Protein kinases play pivotal regulatory roles in most cell communication and metabolic pathways. Thus, new methods for assaying protein kinase activities are not only valuable in the elucidation of various biological events but are also of profound utility in protein-kinase-targeted drug discovery. Although standard radiometric assays and antibody-based enzyme-linked immunosorbent assays (ELISA) detect pro tein kinase activities, these methods measure in vitro protein kinase activities discontinuously using a radiolabeling proce dure and a phosphopeptide-specific antibody treatment, respectively. These methods cannot be applied to protein kinase assays in living cells. Genetically encoded fluorescent protein reporters capable of fluorescent resonance energy transfer (FRET) have been designed to detect protein kinase activities continuously in single living cells. However, these phosphorylation-responsive fluorescent protein reporters exhibit only modest fluorescence changes after protein phosphorylation. Moreover, these methods are limited to cells genetically modified to express fluorescent protein reporters.