Visit our Expo - Redox and Inflammation signaling 2012

Session II : Receptor signaling and G proteins Poster II, 9
Genetically-encoded, FRET-based sensors for monitoring protein kinase signaling
Justin J. Brumbaugh, Christiane Jost, Carsten Schultz
Gene Expression Program, European Molecular Biology Laboratory (EMBL),
Heidelberg 69117, Germany. Email: brumbaug@embl.de, jost@embl.de,
Schultz@embl.de
Protein phosphorylation and dephosphorylation are important forms of posttranslational
regulation and a fundamental means for controlling cellular fate and function. Deviant kinase
and phosphatase activities are responsible for numerous diseases, and in particular, cancer.
Due to the far reaching implications of phosphorylation and dephosphorylation, it is
imperative to develop tools to closely monitor these dynamic processes in real time.
Previously, we reported the construction of a genetically encoded FRET probe for monitoring
protein kinase C (PKC) activity in living cells (Schleifenbaum et al., J. Am. Chem. Soc.,
2004). To help unravel complex and often interdependent, intracellular signaling we are
working to adapt this probe to other kinase specificities. To this end we have developed two
novel protein kinase A (PKA) sensors and, to the best of our knowledge, the first genetically
encoded dual parameter FRET probe that responds independently to PKA and PKC
stimulation with differential output. Using an array-based approach and automated
microscopy, we have also developed a method using in vivo imaging to screen potential
probes under more comparable conditions and with higher throughput. This technology may
lead to more versatile, cell-based assays for kinase activators and inhibitors.
- 136 -