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

Background Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results 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. Conclusion 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.

• Publication year

  2005

• Venue

  BMC Chemical Biology

• Publication date

  2005-07-05

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1186/1472-6769-5-1PMID 15998468PMCID 1180429
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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