- Meeting abstract
- Open Access
Rational design of a PKA-based sensor for cGMP
BMC Pharmacology and Toxicology volume 16, Article number: A64 (2015)
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 . Introduction of these amino acids into PKA switches its cyclic nucleotide selectivity . 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 . 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.
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.
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.
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.
About this article
Cite this article
Lorenz, R., He, D., Kim, C. et al. Rational design of a PKA-based sensor for cGMP. BMC Pharmacol Toxicol 16, A64 (2015). https://doi.org/10.1186/2050-6511-16-S1-A64
- Mouse Embryonic Fibroblast
- Nucleotide Binding Domain
- Aforementioned Mutation
- Homologous Enzyme
- Equal Affinity