A novel Photo Cross Linking Assay (PCLA) for targeting protein phosphorylation
Protein phosphorylation catalyzed by kinases and phosphatases act at critical control points that regulates many cellular processes. This central role makes them an attractive target for identifying largely untapped and important biomarkers for disease diagnostics. Understanding which proteins interact in a given pathway is an important step to aid in the discovery of therapeutics. Despite the development of a variety of innovative new technologies, dissecting the phosphorylation cascades that regulates cellular activity remains elusive due to lack of reagents that can aid in ultrasensitive detection of minute amounts of phosphoproteins. In an effort to address this challenge the Pflum lab has developed a novel approach of specifically tagging phosphoproteins with ATP analogs for subsequent analysis by mass spectrometry, gel electrophoresis, etc.
PCLA was developed to label kinase substrates by photo cross-linking an ATP analog to associated proteins bound in close proximity to the phosphorylation site. The phosphorylation-dependent crosslinking afforded by PCLA has many possible applications. Including:
v Identification of kinase-substrate pairs
v Characterization of phosphorylation-dependent protein-protein interactions.
v Monitoring of phosphorylation-dependent conformational changes in phosphoprotein structure
With a paucity of methods to monitor phosphorylation-dependent interactions, this new approach can be applied broadly to any phosphorylation-mediated pathway of interest. This is a powerful strategy which ensures that all cross-linked proteins are kinase substrates and involved in a kinase cascade pathway.
Brief description of Technology:
Protein kinases accept ATP analogs modified at the gamma-phosphate group with an aryl azide as a co-substrate to generate the “photoactive” molecular probe. In the presence of UV light, the “photoactive” probe will cross-link with its substrate via a nitrene species creating a covalent bond. The tagged protein is subsequently analyzed by gel electrophoresis or mass spectrometry. Since cross-linking requires kinase-catalyzed phosphorylation, the assay is effective for mapping the kinome, identifying proteins associated with the kinase substrate during kinase reaction, and monitoring phosphorylation-dependent changes in protein conformation due to intra-molecular cross-linking. With few methods available to monitor these phosphorylation-dependent interactions, the technology has the potential to be broadly used to characterize kinase signaling cascades.