SEARCH

SEARCH BY CITATION

Keywords:

  • chemical exchange saturation transfer;
  • biosensor;
  • reporter gene;
  • protein kinase A

Abstract

Purpose:

Protein kinases including protein kinase A (PKA) underlie myriad important signaling pathways. The ability to monitor kinase activity in vivo and in real-time with high spatial resolution in genetically specified cellular populations is a yet unmet need, crucial for understanding complex biological systems as well as for preclinical development and screening of novel therapeutics.

Methods:

Using the hypothesis that the natural recognition sequences of protein kinases may be detected using chemical exchange saturation transfer magnetic resonance imaging, we designed a genetically encoded biosensor composed of eight tandem repeats of the peptide LRRASLG, a natural target of PKA.

Results:

This sensor displays a measurable change in chemical exchange saturation transfer signal following phosphorylation by PKA. The natural PKA substrate LRRASLG exhibits a chemical exchange saturation transfer-magnetic resonance imaging contrast at +1.8 and +3.6 ppm, with a >50% change after phosphorylation with minutes-scale temporal resolution. Expression of a synthetic gene encoding eight monomers of LRRASLG yielded two peaks at these chemical exchange saturation transfer frequencies.

Conclusion:

Taken together, these results suggest that this gene may be used to assay PKA levels in a biologically relevant system. Importantly, the design strategy used for this specific sensor may be adapted for a host of clinically interesting protein kinases. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.