Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity

ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK.EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.Traditional methods for studying signal transduction cascades have been based solely on biochemical analysis of whole cell populations and homogenized tissues (e.g. radioassays, western blots, etc.). In addition, in vitro studies have required the use of radioactive isotopes for biochemical characterization of kinases. These methods are time consuming, produce large quantities of radioactive waste, and do not allow for the study of real-time kinase dynamics.Recently, sensors for studying signal molecules in vitro, as well as cascade dynamics in single cells, have been developed utilizing fluorescent proteins and the phenomenon of fluorescence resonance energy transfer (FRET). FRET is a phenomenon by which energy is transferred from one fluorescent molecule to another by way of dipole-dipole interactions during excitation of the donor molecule. FRET efficiency is given by the equation:where R is the donor-acceptor radius and Ro is the radius at which FRET efficiency is 50% (F？rster radius). Small changes in R (1–2？) and small orientation changes between the donor and acceptor fluorophores can dramatically affect the efficiency of FRET, making very small changes in structure easily detectable. The limitation, however, is