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Rational design of a PKA-based sensor for cGMP


The cAMP-dependent protein kinase (PKA) and the cGMP-dependent protein kinase (PKG) are highly homologous enzymes differing in their specificity for cAMP and cGMP, respectively. Recent structure-function studies led to the identification of key residues responsible for cGMP-specificity in PKG [1]. Introduction of these amino acids into PKA switches its cyclic nucleotide selectivity [2]. Here we demonstrate that the same mutations turn a cAMP-specific FRET-based A-kinase activity reporter (AKAR) into a cGMP-specific reporter.

Methods and results

cGMP-specific threonine and arginine residues found in PKG I were introduced into both the N-terminal and C-terminal cyclic nucleotide binding domains (CNB-A and CNB-B) of the PKA regulatory subunit Iα (RIα) using site-directed mutagenesis. The four resulting constructs were: wild type, CNB-A mutant (T192R/A212T), CNB-B mutant (G316R/A336T) and CNB-A/B mutant which has all aforementioned mutations.

While wild type RIα showed a strong selectivity for cAMP vs. cGMP in both in vitro binding and kinase activation studies, either CNB-A or -B mutants bound both nucleotides with equal affinity, resulting in PKA holoenzymes that were activated by both nucleotides equally. The CNB-A/B mutant had a significantly higher affinity for cGMP, and the corresponding PKA holoenzyme was activated selectively by cGMP.

Mouse embryonic fibroblasts derived from a PKA RIα knockout mouse were contransfected with the RIα constructs along with an AKAR to test their responsiveness for the cell-permeant analogs 8-CPT-cAMP and 8-CPT-cGMP, respectively [3]. Our results showed that both the wild type and the CNB-B mutant do not respond to 8-CPT-cGMP. In contrast, the CNB-A mutant showed a phasic response to 8-CPT-cGMP, and the response of the double mutant CNB-A/B sustained longer.


Cyclic nucleotide-dependent regulation of protein kinase activity is an important aspect of eukaryotic signal transduction. The vice versa specificity of PKA and PKG determines the fidelity of cAMP-PKA and cGMP-PKG pathways. We applied our understanding of cyclic nucleotide selectivity to cellular sensor design and showed that mutating four key residues within a cAMP-specific reporter switches it into a cGMP-specific reporter.

Cyclic nucleotide selectivity has apparently evolved through mutations in the CNB. The identification of these key residues will improve the design of cyclic nucleotide-selective cellular reporters.


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Correspondence to Robin Lorenz.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Lorenz, R., He, D., Kim, C. et al. Rational design of a PKA-based sensor for cGMP. BMC Pharmacol Toxicol 16 (Suppl 1), A64 (2015).

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