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Rational design of a PKA-based sensor for cGMP

Background

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.

Conclusion

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.

References

  1. Huang GY, Kim JJ, Reger AS, Lorenz R, Moon EW, Zhao C, et al: Structural Basis for Cyclic-Nucleotide Selectivity and cGMP-Selective Activation of PKG I. Structure. 2014, 22 (1): 116-124. 10.1016/j.str.2013.09.021.

    Article  CAS  PubMed  Google Scholar 

  2. Lorenz R, Moon EW, Huang GY, Reger AS, Kim JJ, Franz E, Bertinetti D, Kim C, Herberg FW: Transforming PKA into PKG – a structure-function approach to understand cyclic nucleotide selectivity. BMC Pharmacology and Toxicology. 2013, 14 (Suppl 1): P41-10.1186/2050-6511-14-S1-P41.

    PubMed Central  Google Scholar 

  3. Allen MD, Zhang J: Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun. 2006, 348 (2): 716-721. 10.1016/j.bbrc.2006.07.136.

    Article  CAS  PubMed  Google Scholar 

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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 (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) 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). https://doi.org/10.1186/2050-6511-16-S1-A64

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  • DOI: https://doi.org/10.1186/2050-6511-16-S1-A64

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