Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine

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Switching cyclic nucleotide-selective activation of PKA holoenzyme reveals distinct roles of tandem CNB domains. Daniel He, Robin Lorenz, Choel Kim, Friedrich W. Herberg, and Chinten James Lim ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00732 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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ACS Chemical Biology

Switching cyclic nucleotide-selective activation of PKA holoenzyme reveals distinct roles of tandem CNB domains. Daniel He1,#, Robin Lorenz2,#, Choel Kim3, Friedrich W. Herberg2,*, and Chinten James Lim1,* #

Equal contribution

1

Dept. of Pediatrics, University of British Columbia, and Michael Cuccione Childhood Cancer Research Program, BC Children’s Hospital Research Institute, Vancouver, British Columbia, Canada 2 Department of Biochemistry, University of Kassel, Kassel, Hesse, Germany 3 Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA *

Correspondence:

Chinten James Lim, [email protected] Department of Pediatrics, University of British Columbia, 3092-950 West 28th Ave., Vancouver, B.C., Canada, Tel.: (604) 875-2000x4795; Fax: (604) 875-3120; E-mail: [email protected] Friedrich W. Herberg, [email protected] Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, Tel.: (+49)561-8044511; E-mail: [email protected] Running title: Cyclic Nucleotide Selectivity of PKA Regulatory Subunit Key words: cAMP; cGMP; PKA activation; PKG; FRET Abbreviations 6-AH-cAMP, N6-(6-Aminohexyl)adenosine-3', 5'-cyclic monophosphate; 8-AHA-cAMP, 8-(6Aminohexylamino)adenosine-3',5'-cyclic monophosphate; 8-CPT-cAMP, 8-(4chlorophenylthio)adenosine 3′,5′-cyclic monophosphate; 8-CPT-cGMP, 8-(4chlorophenylthio)guanosine 3′,5′-cyclic monophosphate; AKAP, A-kinase anchoring protein; C, catalytic; cAMP, cyclic AMP; cGMP, cyclic GMP; CAP, catabolite activator protein; CNB, cyclic nucleotide-binding; CFCA, calibration-free concentration analysis; CNB-A, N-terminal cyclic nucleotide-binding domain; CNB-B, C-terminal cyclic nucleotide-binding domain; EC50, half maximal effective concentration; FRET, Fluorescence Resonance Energy Transfer; GKIP, G-kinase interacting protein; IBMX, 3-isobutyl-1-methylxanthine; Kact, activation constants; MEF, mouse embryonic fibroblast; NO, nitric oxide; PBC, phosphate binding cassette; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKI, heat-stable protein kinase inhibitor; R, regulatory; RIαmutCNB-A, regulatory subunit Iα T192R A212T; RIαmutCNB-B, regulatory subunit Iα G316R A336T; RIαmutCNB-A,B, regulatory subunit Iα T192R A212T G316R A336T; RIαwt, wild type regulatory subunit Iα; SEM, standard error of the mean; SPR, surface plasmon resonance 1 ACS Paragon Plus Environment


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ABSTRACT

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are key effectors of cyclic nucleotide signaling. Both share structural features that include tandem cyclic nucleotide-binding (CNB) domains, CNB-A and CNB-B, yet their functions are separated through preferential activation by either cAMP or cGMP. Based on structural studies and modeling, key CNB contact residues have been identified for both kinases. In this study, we explored the requirements for conversion of PKA activation from cAMP-dependent to cGMP-dependent. The consequences of the residue substitutions T192R/A212T within CNB-A and/or G316R/A336T within CNB-B of PKA-RIα on cyclic nucleotide binding and holoenzyme activation were assessed in vitro using purified recombinant proteins, and ex vivo using RIα-deficient mouse embryonic fibroblasts genetically reconstituted with wild type or mutant PKA-RIα. In vitro, a loss of binding and activation selectivity was observed when residues in either one of the CNB domains were mutated, while mutations in both CNB domains resulted in a complete switch of selectivity from cAMP to cGMP. The switch in selectivity was also recapitulated ex vivo, confirming their functional roles in cells. Our results highlight the importance of key cyclic nucleotide contacts within each CNB domain and suggest that these domains may have evolved from an ancestral gene product to yield two distinct cyclic nucleotide-dependent protein kinases.

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INTRODUCTION

Cyclic AMP-dependent protein kinase (PKA; Protein Kinase A) and cyclic GMP-dependent protein kinase (PKG; Protein Kinase G) are structurally related but functionally distinct kinases within the AGC family of serine/threonine protein kinases (Figure 1A).1 In its inactive state, PKA forms a tetrameric holoenzyme complex consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits.2 One R-subunit inhibits one C-subunit by binding to the active site cleft, and the two R-subunits are bound together via an N-terminal dimerization domain. In contrast to the heterotetrameric conformation of PKA, PKG exists as a homodimer of two identical subunits each containing both the R- and C-domains fused together on a single polypeptide chain.3 Furthermore, PKA and PKG are similar in that they are both activated by cyclic nucleotide binding to their respective R-domains, which contain cyclic nucleotide binding (CNB) domains related to the bacterial catabolite gene activator protein (CAP).4, 5 Both kinases can be activated by cAMP and cGMP, respectively; however, while PKA is activated by considerably lower concentrations of cAMP, PKG is preferentially activated by cGMP.6-9 Isolated CNB domains have been shown to be cyclic nucleotide-selective and this selectivity is thought to keep both signaling pathways segregated for eliciting specific responses.10-12 In addition to pools of cyclic nucleotides, further intracellular segregation of signaling involves pathway-specific coupling between kinases and substrates through A-kinase anchoring proteins (AKAPs) for PKA and G-kinase interacting proteins (GKIPs) for PKG.13, 14

PKA is part of many signal transduction pathways governing cellular processes such as migration, apoptosis, and immunity.15 Different isotypes of PKA holoenzymes (as defined by the R-subunits: RIα, RIβ, RIIα or RIIβ) can be targeted to subcellular compartments by a large repertoire of AKAPs or show tissue-specific expression. The RIα holoenzyme can be thought of as the master regulator of intracellular PKA activity and, as such, is located ubiquitously throughout the cell.16 While RIIα knockout mice have no discernible physiological defects other than increased sensitivity to AKAP inhibitors,17 the embryonic lethality of RIα knockout mice highlights the importance of the RIα subunit in the cAMP-mediated regulation of PKA activity.18 As a master regulator, RIα expression levels can increase to compensate for loss of the other Rsubunits, while loss of RIα is not adequately compensated for by the remaining R-subunits 3 ACS Paragon Plus Environment


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resulting in increased basal PKA activity due to excess free C-subunits.17-20 Additionally, mutations leading to RIα haploinsufficiency result in Carney complex, a disorder characterized by the formation of myxomas.21 Mutations within the CNB domains of RIα can lead to the phenotypically different Carney complex or acrodysostosis, which can be traced to functional differences in PKA activity that is increased or constitutive for Carney complex, and decreased for acrodysostosis.22

While PKA has a broad range of functions, PKG is a key kinase in the nitric oxide (NO)/cGMP signaling pathway and serves to promote platelet disaggregation, smooth muscle relaxation, and vasodilation.14 There are two types of PKG, I and II. Type I PKG is the main regulator of smooth muscle tone and knockout mice exhibit hypertension and defects in smooth muscle contraction.23 On the other hand, type II PKG knockout mice show deficiencies in intestinal fluid secretion as well as dwarfism due to reduced bone growth.24

Since activation of both PKA and PKG is mediated by cyclic nucleotides, the binding selectivity of cyclic nucleotides by the R-domains is a crucial mechanism in segregating their respective signaling pathways. Although previous studies have identified T193, T317, and R297 of human PKG Iβ as key residues for selective binding of cGMP,8, 11, 25, 26 their specific roles in cyclic nucleotide selectivity have yet to be investigated within a cellular setting. Here, we present a novel system utilizing Fluorescence Resonance Energy Transfer (FRET) microscopy and RIαdeficient mouse embryonic fibroblasts reconstituted with wild type (WT) RIα or cyclic nucleotide binding mutants to examine the molecular mechanisms of cyclic nucleotide selectivity as well as PKA activation. The ex vivo (in-cell) activities are contrasted and compared to the activities observed for purified recombinant proteins in vitro. In vitro, we found that mutation of either CNB domain abolished the cyclic nucleotide selectivity, while the combined mutations in both CNB-A and CNB-B switched the cyclic nucleotide selectivity from cAMP to cGMP in binding and activation. When reconstituted ex vivo as functional PKA holoenzymes bearing the wild type and mutant RIα subunits, we also found that the combined CNB-A and CNB-B mutations effectively switched cyclic nucleotide dependent-activation of the kinase from a cell permeable cAMP-analog to its cGMP counterpart. In addition, the ex vivo PKA holoenzyme assays implicate the importance of the cyclic nucleotide selectivity of CNB-A over that of CNB4 ACS Paragon Plus Environment

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B. Our findings underline the relevance of the key residues introduced for cGMP specificity and imply a sequence divergence of the PKA and PKG CNB domains in evolution.



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METHODS

Reagents The cyclic nucleotide analogs 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8CPT-cAMP) and 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (8-CPT-cGMP) (Sigma-Aldrich) were solubilized in H2O at a stock concentration of 20 mM. Cells were stimulated with cyclic nucleotide analogs at a final concentration of 500 µM.

Plasmid construction The FRET-based PKA activity biosensor construct NES-AKAR3 was a generous gift from J. Zhang (Johns Hopkins University).27 The construct expressing wild type human RIα is as described previously.28 Cyclic nucleotide binding domain mutations were generated by sitedirected mutagenesis using the Stratagene QuikChange methodology. Mutations in the CNB-A domain were made using oligos corresponding to the sequences: 5'GTTAACAATGAATGGGCAAGGAGTGTTGGGGAAGGAGG-3' for T192R and 5'TTTATGGAACACCGAGAACAGCCACTGTCAAAGCA-3' for A212T. Mutations in the CNB-B were made with: 5'-AAGAGTTTGTTGAAGTGCGAAGATTGGGGCCTTCT-3' for G316R and 5'-GATGAATCGTCCTCGTACTGCCACAGTTGTTGC-3' for A336T. All constructs were sequenced to verify introduction of the desired mutations. Finally, all RIα constructs were subcloned as a C-terminal fusion to mCherry in a pcDNA3 (Life Technologies) vector backbone. For recombinant expression in E. coli, the human RIα constructs were subcloned into the pQTEV plasmid via BamHI and HindIII restriction sites using the primers 5’TGGATCCATGGAGTCTGGCAG-3’ and 5’-AGCAAGCTTTCAGACAGACAGTGAC-3’. The expressed proteins contain an N-terminal 7xHis-tag and a tobacco etch virus (TEV) protease cleavage site.29

Cell culture and transfection Cell experiments were conducted using prkar1a-/- mouse embryonic fibroblasts (herein referred to as RIα-/- MEF)18, 28 cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), non-essential amino acids (NEAA, Life Technologies) and penicillin– 6 ACS Paragon Plus Environment

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streptomycin (Life Technologies). Cells were incubated at 37°C with 5% CO2 and kept between passages 2 and 20. RIα-/- MEF cells were transiently transfected by nucleoporation using the Amaxa Nucleofector 2b Device (program A-023) and left to grow overnight prior to cell imaging studies. In additional experiments, lipid-based transient transfection was carried out using Lipofectamine 2000 (ThermoFisher).

Protein expression and purification The human RIα constructs were expressed in E. coli ∆cya TP2000 cells, which lack adenylyl cyclase activity.30, 31 The inoculated liter cultures were incubated at 37°C with shaking at 180 rpm until growth reaching an optical density of ~0.6 to 0.8, following which 400 µM IPTG was added to induce protein expression overnight at room temperature. Cells were harvested and stored at -20°C.

For purification, the cells were resuspended in lysis buffer containing 50 mM KH2PO4, pH 8.0, 500 mM NaCl, 20 mM imidazole and 5 mM 2-mercaptoethanol with complete™ EDTA-free protease inhibitor (Sigma-Aldrich), 1 mM PMSF and 0.1 mg/mL lysozyme. The homogenate was passed three times through a French pressure cell press (Thermo Electron Corp.) at 16,000 psi. The cell debris was spun down at 45,000xg for 45 min and the supernatant was loaded onto a 1 mL Protino Ni-NTA column (Macherey-Nagel) using an ÄKTApurifier UPC 10 machine (GE Healthcare). The His-tagged protein was eluted with lysis buffer containing 250 mM imidazole. Elution fractions were checked on SDS-PAGE gels.32 The proteins were further purified by anion exchange chromatography using a 1 mL ResourceQ column or by gel filtration using a 16/60 Superdex 75 column. RIα constructs were stored at -20°C in 20 mM MOPS, pH 7, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA and 5 mM 2-mercaptoethanol.

Human PKA-Cα was expressed in E. coli BL21 (DE3) cells and purified via an PKI5-24-resin as previously described.33, 34

Cell imaging RIα-/- MEF cells transfected to express mCherry-RIα and NES-AKAR3 were replated on fibronectin-coated coverslips in serum-reduced DMEM (1% FBS) for 2 hours prior to imaging. 7 ACS Paragon Plus Environment


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Live cell FRET-imaging was performed on an Olympus IX-81 microscope equipped with an active focus system, X-Cite exacte light source (EXFO), Proscan II stage (Prior Scientific), CoolSnap HQ2 camera (Photometrics), Metamorph software controller (Molecular Devices) and environmental incubation (37°C, 5% CO2 and humidity). FRET fluorescence microscopy images were acquired using the 60X NA 1.35 oil objective, and were taken at defined intervals at 300 ms exposure before and after application of cNMP stimuli for up to 30 minutes. CFP (480/30 nm) and YFP (535/40 nm) emission stimulated by CFP excitation (438/24 nm) were captured simultaneously using an image splitter (Photometrics DV2) equipped with 505nm dichroic mirror (Chroma). Imaging of mCherry was accomplished with 589/15 nm exciter and 632/22 nm emitter (Semrock). Only cells expressing comparable fluorescence for mCherry-RIα and NESAKAR3 were selected for imaging. Post-acquisition ratiometric image analysis was carried out on ImageJ with the Ratio Plus plugin as previously published.35 The YFP/CFP FRET ratiometric value for each cell at each time point was calculated as the average values of multiple ROIs (region of interest) within the cytoplasm. FRET values were further normalized against the average FRET values obtained before stimulation with cyclic nucleotides. Box-whisker plots of the analyzed data (20-26 cells per condition) were generated with ggplot2.36 For imaging of immunofluorescence stained RIα-/- MEF cells expressing mCherry-RIα, cells adherent on fibronectin-coated coverslips were fixed using 3.7% formaldehyde diluted in modified TBS (50 mM Tris, 100 mM NaCl, 20 mM Na4P2O7, 2 mM NaF, pH 7.4). Cells were permeabilized with 0.1% Triton X-100, blocked with 2% BSA/2% NGS (normal goat serum), stained with α-phospho-(S/T) PKA substrate antibody (Cell Signaling Technology) and DyLight 488 conjugated goat anti-rabbit antibody (Pierce). Reagent dilution and all washes were performed in TBS. Labeled cells were mounted with ProLong Gold (Life Technologies). Widefield epi-fluorescence images were acquired using the 40X NA 0.75 objective on the Olympus IX-81 for phospho-PKA substrate staining (FITC channel) and mCherry-RIα (mCherry channel). To minimize signal intensity variance resulting from different focal distances, all images were acquired using an identical (or fixed) focus offset. For analysis, the mean fluorescence for phospho-PKA substrate staining for each cell was computed from 4 ROIs distributed within the extra-nuclear cytoplasm. ‘Relative PKA Activity’ was then computed as the mean phospho-PKA substrate signal obtained for each mCherry-RIα transfected cell divided 8 ACS Paragon Plus Environment

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by the average obtained from 3 non-transfected reference cells within the same field of view. Scatter plots of the analyzed data were generated using GraphPad Prism.

Surface Plasmon Resonance (SPR) All SPR experiments were performed on a Biacore T100/T200 machine (GE Healthcare) with 20 mM MOPS, 150 mM NaCl and 0.05% P20 surfactant as running buffer. The cAMP analogs 8(6-Aminohexylamino)adenosine-3',5'-cyclic monophosphate (8-AHA-cAMP), N6-(6Aminohexyl)adenosine-3', 5'-cyclic monophosphate (6-AH-cAMP) and 2-(6Aminohexylamino)adenosine-3', 5'- cyclic monophosphate (2-AHA-cAMP) (BioLog Life Science Institute) were coupled to high density to flow cells 2, 3 and 4, respectively, of an Sseries CM5 sensor chip using amine coupling as previously described.37 Flow cell 1 was activated and deactivated and used as a reference surface. For solution competition experiments,38 the RIα proteins were pre-incubated with various concentrations of cAMP or cGMP, respectively, and injected over all sensor chip surfaces for 150 to 900 s. The dissociation phase was monitored for 75 s. The SPR signal at the beginning of the dissociation phase was plotted against the logarithmic competitor concentration and half maximal effective concentrations (EC50) were calculated from sigmoidal dose-response curves using GraphPad Prism 6.01. At the end of each cycle, the surfaces were regenerated by injecting 0.5% SDS and 1 M NaCl for 60 s each. All sensorgrams depicted were double-referenced by subtracting buffer injection signals and the signals of the reference flow cell. To determine the active protein concentration with regard to cyclic nucleotide binding, calibration-free concentration analysis (CFCA), implemented in the Biacore T100/200 control software, was applied using high-density cAMP analog chips as described in the Biacore T100 software package.

Spectrophotometric kinase activity assay PKA holoenzymes were formed by mixing human PKA-Cα with the respective RIα proteins at a 1:1.2 molar ratio and by subsequent dialysis against 20 mM MOPS, 150 mM NaCl, 10 mM MgCl2, 1 mM ATP and 5 mM 2-mercaptoethanol overnight. Kinase activity was measured using an enzyme-coupled assay according to Cook et al.39. To measure the cyclic nucleotide-dependent 9 ACS Paragon Plus Environment


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kinase activation, the respective PKA holoenzyme was pre-incubated with variable concentrations of cAMP or cGMP, respectively, in assay mix (100 mM MOPS, pH 7, 10 mM MgCl2, 1 mM phosphoenolpyruvate, 1 mM ATP, 150 U lactate dehydrogenase, 84 U pyruvate kinase, 220 µM NADH and 5 mM β-mercaptoethanol) and the reaction was started by adding 250 µM Kemptide substrate peptide (LRRASLG). The absorbance at 340 nm was followed for 1 min. Phosphotransferase activity was plotted against the logarithmic cyclic nucleotide concentration. Activation constants (Kact) were determined from sigmoidal dose-response curves using GraphPad Prism 6.01.


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RESULTS and DISCUSSION

Cyclic nucleotide binding selectivity of WT and mutant RIα. While PKA I and PKG I share conserved CNB domains, we previously showed that PKG I displays additional amino acid contacts that confer its cGMP specificity.11, 25 These contacts are conserved threonine or serine residues (T193 in CNB-A or T317 in CNB-B of PKG Iβ) in the phosphate binding cassette (PBC) and an arginine residue (R297 in CNB-B) at β5 that specifically interact with the guanine moiety. To determine if introducing these contacts into RIα switches its cyclic nucleotide binding specificity, we mutated the analogous residues in the CNB domains of RIα to threonine and arginine (T192R/A212T in CNB-A and G316R/A336T in CNBB, respectively) (Figure 1). In sum, we generated three mutants of RIα with these mutations engineered into only the CNB-A or CNB-B domains (RIαmutCNB-A or RIαmutCNB-B) or both (RIαmutCNB-A,B) and compared their affinities for either cAMP or cGMP (EC50), and their activation constants (Kact). The cyclic nucleotide binding affinities of the wild type and mutant RIα subunits were determined using Surface Plasmon Resonance (SPR) competition experiments (Figure 2 and Table 1). As reported previously,10 the EC50 cGMP/cAMP ratio for RIαwt was about 500-fold, indicating a high binding selectivity for cAMP over cGMP. Compared to RIαwt, RIαmutCNB-A exhibited a 1000-fold decrease in the EC50 for cGMP while the affinity for cAMP was unaffected, resulting in a weak cGMP-binding specificity. For RIαmutCNB-B, a 50-fold decrease in the cGMP EC50 was observed. The cGMP/cAMP EC50 ratio of the RIαmutCNB-B subunit was about 16, implying a reduced selectivity for binding cAMP over cGMP compared to RIαwt. Strikingly, the quadruple mutant RIαmutCNB-A,B showed a 12-fold increase in the cAMP EC50 and a >3800fold decrease in the cGMP EC50, with a cGMP/cAMP EC50 ratio of 0.01, indicating a switched cyclic nucleotide binding selectivity from cAMP to cGMP as compared to RIαwt (Table 1).

Cyclic nucleotide activation of PKA with WT and mutant RIα subunits. To investigate the effects of altered cyclic nucleotide binding on PKA activation, activation constants (Kact) for either cAMP or cGMP were measured (Figure 3). The RIαwt holoenzyme was specifically activated by cAMP (Kact 0.053 µM) with a more than 100-fold preference in 11 ACS Paragon Plus Environment


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comparison to cGMP (Kact 7.4 µM), which is in good agreement with previous studies.8, 40 RIαmutCNB-A showed Kact values of 0.46 µM for cGMP and 0.36 µM for cAMP, indicating a loss of cAMP selectivity. As for RIαmutCNB-B, the Kact of cGMP was comparable to that of RIαmutCNB-A (0.54 µM versus 0.46 µM), whereas the cAMP Kact was 3-fold lower (0.11 µM versus 0.36 µM). Unlike RIαmutCNB-A, RIαmutCNB-B is slightly more selective for cAMP in activation. Finally, RIαmutCNB-A,B showed Kact values of 0.048 µM for cGMP and 0.80 µM for cAMP, reversing its selectivity for cAMP over cGMP (Table 2). All RIαwt and mutant holoenzymes form stable complexes in the absence of cNMPs and are fully activated by cAMP and cGMP (Supplementary Figure 1). Our work complements and extends previous studies by Shabb et al 8, 26 with additional mutations introduced in RIα that not only increases its affinity for cGMP, but also decreases its affinity for cAMP. Previous studies showed that the A212T and A336T substitutions in CNB-A and CNB-B of RIα, respectively, produced PKA with increased affinity for cGMP, but with little change in cAMP affinity.8 Our previous studies had revealed additional cyclic nucleotide contact residues, in this case, R297 in CNB-B of PKG that specifically interacts with the guanine moiety of cGMP.11, 25 This structure-informed insight allowed us to introduce a specific arginine in conjunction with A212T and A336T, as quadruple mutations T192R, A212T, G316R and A336T, to obtain type I PKA exhibiting both an increased affinity for cGMP binding and a decreased affinity for cAMP binding. Thus, we were able to switch the cyclic nucleotide selectivity in favor of cGMP 50-fold over cAMP. As both T192R and G316R mutations are based on the R297 residue of the PKG I CNB-B domain (Figure 1D), our results confirm the critical role of R297 in achieving high cGMP selectivity.11

The data obtained from the cyclic nucleotide binding selectivity and PKA activation studies reveal different characteristics of the CNB domains. The cAMP affinity of RIα remained unchanged when either CNB-A or CNB-B was mutated. However, mutating both CNB domains at a time reduced the affinity for cAMP. This suggests that in the single domain mutants, the respective wild type domain compensates for the mutated domain. Mutation of the CNB-A more strongly increased the affinity for cGMP than mutation of the CNB-B which implies that CNB-A has a stronger impact on binding selectivity than CNB-B. Mutation of the RIα CNB-A domain 12 ACS Paragon Plus Environment

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resulted in a loss of selectivity between cAMP and cGMP, which was corroborated with the PKA activation assay. In contrast to the binding data, mutation of CNB-A alone increased the Kact for cAMP in comparison to the wild type, underlining the role of the CNB-A as an allosteric switch. CNB-A can switch between two conformational changes, either binding and thereby inhibiting the C-subunit, or binding cAMP.41, 42 In the inactive PKA holoenzyme, the CNB-As are thought to be masked by the C-subunits and only become accessible when the CNB-Bs are occupied. The RIαmutCNB-B showed a 16-fold preference for binding cAMP over cGMP (albeit reduced in comparison to RIαwt); however, this drastic difference was not seen in the PKA activation assay, as the RIαmutCNB-B cGMP Kact was only 5-fold higher than the cAMP Kact, and 14-fold lower compared to the RIαwt cGMP Kact. Finally, mutating both CNB-A and CNB-B (RIαmutCNB-A,B) dramatically switches its selectivity in binding and activation, turning PKA from a cAMPdependent to a cGMP-dependent protein kinase. Reconstitution of WT and mutant RIα holoenzymes in RIα-/- cells. To examine the altered cyclic nucleotide selectivity of RIα upon mutations in a cellular context, we transfected the wild type and mutant RIα as mCherry-tagged fusion proteins into MEF cells lacking RIα expression (RIα-/-), and correspondingly, lacking type I PKA holoenzyme. Compared to WT fibroblasts, RIα-/- cells have excess unbound and therefore active catalytic subunits, resulting in increased PKA activity even under non-stimulated conditions.18, 28 This increased activity can be inhibited with the small molecule, H89, or by transfecting RIα-/- cells to express the specific PKA inhibitor, PKI (as mCherry-PKI) (Supplementary Figure 2). To confirm reconstitution of the PKA holoenzyme bearing the expressed mCherry-RIα, transfected RIα-/MEFs were fixed and stained using an antibody directed against substrates phosphorylated by PKA, as readout for PKA activity (Figure 4A, Supplementary Figure 2). When compared to nontransfected RIα-/- cells, expression of each of the four RIα constructs resulted in cells with significantly reduced staining for PKA-phosphorylated substrates, an indication of lower PKA activity under non-stimulated conditions (Figure 4B). Therefore, transfection of the mCherrytagged wild type and mutant RIα subunits into RIα-/- cells successfully reconstituted the PKA holoenzyme as indicated by decreased basal phosphorylation of PKA substrates.

Intracellular activation of WT and mutant RIα holoenzymes. 13 ACS Paragon Plus Environment


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To examine the cyclic nucleotide stimulation properties of the RIα CNB-A and CNB-B mutants in live cells, we co-transfected RIα-/- MEFs with the mCherry-tagged RIα constructs and a genetically encoded FRET-based biosensor for PKA activity termed AKAR (A-kinase activity reporter). Briefly, the expressed AKAR polypeptide contains a PKA substrate motif and a phosphoamino acid binding domain that is flanked by yellow (YFP) and cyan (CFP) fluorescent proteins. When the substrate site is phosphorylated by PKA, AKAR undergoes a conformational change bringing the YFP and CFP proteins in close proximity that can be visualized as increased YFP/CFP emission ratio upon CFP excitation.35 For the purposes of our investigations, we used the cytosolic targeted AKAR (NES-AKAR), as changes in cytosolic PKA activity tended to be more robust compared to that found in the nucleus or at the plasma membrane.27

First, we treated transfected cells with the cell permeable cAMP analog, 8-CPT-cAMP, and monitored PKA activity as changes in YFP/CFP fluorescence emission ratio (Figure 5). Cells expressing mCherry-RIαwt exhibited measurable increases in PKA-mediated FRET within 20 minutes following 8-CPT-cAMP addition (Figure 5A,5C and Supplementary Table 1). We noted that approximately half of cells expressing mCherry-RIαmutCNB-B exhibited a measurable response to 8-CPT-cAMP. In contrast, the vast majority of cells (>75%) expressing mCherry-RIαmutCNB-A or mCherry-RIαmutCNB-A,B exhibited minimal changes in FRET activity when stimulated with 8CPT-cAMP, suggesting that mutations introduced within CNB-A of RIα greatly diminished the ability of 8-CPT-cAMP to activate PKA in cells.

Next, we utilized the same analyses to monitor PKA FRET activity for transfected cells treated with the cell permeable cGMP analog, 8-CPT-cGMP. The vast majority of cells (>75%) expressing mCherry-RIαwt or mCherry-RIαmutCNB-B exhibited minimal PKA-mediated FRET activity upon stimulation with 8-CPT-cGMP (Figure 5B, 5C and Supplementary Table 1). In contrast, the majority of cells expressing mCherry-RIαmutCNB-A,B, and approximately half of the cells expressing mCherry-RIαmutCNB-A, were positively stimulated with 8-CPT-cGMP. Taken together, the results obtained from ex vivo cellular PKA activation studies indicate that the mutations introduced within CNB-A favor activation by cGMP while diminishing activity mediated by cAMP. Strikingly, combining mutations in both CNB domains (RIαmutCNB-A,B) resulted in robust conversion of PKA holoenzymes from cAMP- to cGMP-dependent. 14 ACS Paragon Plus Environment

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Overall, our in-cell stimulations of RIα constructs with 8-CPT-cAMP and 8-CPT-cGMP had opposing results, as each RIα construct showed PKA activation in response to one of the cyclic nucleotides but not the other. The factor determining activation to a specific cyclic nucleotide appears to be the binding selectivity of CNB-A (shown as a schematic in Figure 6). Both RIαwt and RIαmutCNB-B possessed the wild type CNB-A, and both resulted in PKA activation upon 8CPT-cAMP stimulation. Likewise, both RIαmutCNB-A and RIαmutCNB-A,B possessed the mutated CNB-A, and stimulation of both with 8-CPT-cGMP resulted in PKA activation. In our in vitro studies investigating the purified recombinant RIαmutCNB-A protein, we found a loss of cAMP/cGMP selectivity for nucleotide binding as well as PKA activation. RIαmutCNB-B still favored cAMP, but its selectivity was reduced compared to RIαwt, and a similar reduction was seen in PKA activation. Similarly, we observed a loss of cAMP/cGMP selectivity in PKA activation for both RIαmutCNB-A and RIαmutCNB-B constructs in our ex vivo experiments. It is evident that binding of cyclic nucleotides by both CNB domains is required for robust activation of PKA. However, stimulations of RIα-/- cells with cyclic nucleotide analogs were at least partially successful in eliciting activation of PKA when the RIα construct contained CNB-A that was predicted to bind the selected cyclic nucleotide. This underlines the idea that full activation of PKA ex vivo is largely dependent on cyclic nucleotide binding to CNB-A, as has been shown in previous studies.43, 44 Furthermore, the cyclic nucleotide selectivity of CNB-B seemed to have little to no effect on PKA activation ex vivo under the conditions used in this study. In isolation, CNB-B of RIα is more selective for cAMP compared to CNB-A.10 The differences observed for the full-length RIα may be influenced by cooperative binding of cyclic nucleotides, and by interactions involving the catalytic subunit.44 Within the PKA holoenzyme complex, CNB-A is masked by the catalytic subunit, while cAMP binding to CNB-B is essential for cAMP binding to CNB-A. These dynamics may be altered when the affinity of the cyclic nucleotide to CNB-A is sufficient to bypass the requirement of cyclic nucleotide binding to CNB-B. The results are in line with the previously proposed model of PKA activation, as CNB-A is the determining step in eliciting full activation.45 An interesting contrast is seen in PKG Iβ, where



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CNB-A lacks cAMP/cGMP selectivity and the CNB-B regulates activation by being highly selective for cGMP.11, 25, 46

Conclusions Results presented here suggest that PKA and PKG have evolved from an ancestral gene product that developed cAMP- or cGMP-specificity through single amino acid changes. The resulting differences in the primary structures of both kinases enable the segregation of cAMP- and cGMP-signaling pathways. Still, both PKA and PKG seem to be regulated by a number of cyclic nucleotide species, thereby integrating several pathways through cross-talk.47 Along these lines, both cAMP and cGMP regulate the activation of both kinases.48-50 Based on the sequence homology of the CNB-A and CNB-B domains of PKA and PKG, it can be postulated that the tandem CNB domains may have evolved from an ancestral gene duplication event.51, 52 Interestingly, up to now, no cyclic nucleotide effector protein has been described to have one cAMP-specific CNB-domain and one cGMP-specific CNB domain. Such a hybrid would possibly have only a marginal preference for binding as well as activation by one of the cyclic nucleotides. It is intriguing to consider that single nucleotide exchanges in two amino acid residues implicated in this study (RIα A212 codon is GCA, PKG Iβ T193 codon is ACA; RIα G316 codon is GGA, PKG Iβ R297 codon is AGA) may form the evolutionary basis for cAMP vs cGMP selectivity of the CNB domains.5, 26 ACKNOWLEDGEMENTS C.J.L. is supported by grants from NSERC (418320-2012), CIHR (MOP-137033) and CFI (25036). C.J.L. and D.H. acknowledge the support of the Michael Cuccione Foundation. F.W.H. is funded by the Deutsche Forschungsgemeinschaft (DFG HE 1818/10) and by the Future-project Phosmorg, University of Kassel. R.L was supported by a Ph.D. fellowship of the University of Kassel as a member of the graduate program Functionomics. F.W.H. and R.L. acknowledge the Center for Interdisciplinary Nanostructure Science and Technology (CINSaT) of the University of Kassel for support of this work. C.K. is supported by National Institutes of Health (NIH) grant R01 GM090161. We thank D. Bertinetti and S. Schmidt for helpful discussions, and M. Hansch and O. Bertinetti for expert technical assistance.


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Supporting Information Available: Supplementary table 1, supplementary figure 1 and supplementary figure 2. This material is available free of charge via the internet at http://pubs.acs.org.

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Table 1 Binding data of RIα CNB domain mutants. EC50 values for cAMP and cGMP are derived from SPR solution competition experiments. Values were calculated from sigmoidal dose response curves as shown in Fig. 2. n: Number of measurements from two independent protein preparations. The EC50 ratio was calculated as a measure of selectivity. PKA R-subunit RIαwt RIαmutCNB-A RIαmutCNB-B RIαmutCNB-A,B

EC50 ± SEM (n) cAMP cGMP 3.2 ± 0.5 nM (13) 1700 ± 300 nM (8) 5.7 ± 1 nM (12) 1.7 ± 0.40 nM (9) 2.1 ± 0.3 nM (12) 33 ± 8 nM (13) 38 ± 4 nM (9) 0.44 ± 0.05 nM (15)

EC50 cGMP / EC50 cAMP 530 0.3 16 0.012

Table 2 Activation data of RIα CNB domain mutants. Kact values for cAMP and cGMP were calculated from sigmoidal dose response curves (activation curves) as shown in Fig. 3. n: Number of measurements from two independent protein preparations. Kact ratio was calculated as a measure of selectivity.

PKA R-subunit RIαwt RIαmutCNB-A RIαmutCNB-B RIαmutCNB-A,B

Kact ± SEM (n) cAMP 0.053 ± 0.02 µM (2) 0.36 ± 0.06 µM (2) 0.11 ± 0.01 µM (2) 0.80 ± 0.27 µM (2)

cGMP 7.4 ± 2.2 µM (2) 0.46 ± 0.08 µM (2) 0.54 ± 0.15 µM (2) 0.048 ± 0.01 µM (2)

Kact cGMP / Kact

cAMP 140 1.27 5.04 0.06


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Figure 1 Schematics of PKA and PKG with emphasis on the cyclic nucleotide binding domains. (A) Both PKA and PKG contain dimerization/docking (D/D) domains that bind to anchoring proteins such as AKAPs and GKIPs for localization, inhibitor sites to bind to the active site cleft of the catalytic subunit, and two cyclic nucleotide binding domains, CNB-A and CNB-B. The catalytic subunits are well conserved amongst protein kinases, consisting of a N-terminal lobe for ATP binding and a C-terminal lobe for catalytic activity. While PKA exists in a tetrameric structure, PKG is a dimer, with both monomers containing a regulatory and a catalytic domain. (B) Structures of RIα complexed with cAMP and PKG Iβ complexed with cGMP are shown on the left (PDB codes 1RGS and 4Z07) and their alignments of each CNB domain on the right. In the left panel, the CNB-A domains are colored in magenta, CNB-B in teal, and PBC (phosphate binding cassette) in yellow. Cyclic GMPs are shown as stick and colored by atom type (carbon, white; nitrogen, blue; oxygen, red; and phosphorus, orange). The Cα atoms of residues important in this study are shown with red spheres. In the right panels, the CNB domains of PKG Iβ are shown in gray. (C) Structural models of CNB domain mutants. The models were generated by T192R, A212T substitutions in CNB-A, and G316R, A336T substitutions in CNB-B using the crystal structure of RIα as a template (PDB ID 1RGS). Key cGMP contact residues are shown as stick and hydrogen bonding interactions with cGMP are shown as dotted lines. (D) Sequence alignment of the CNB-As and CNB-Bs of RIα and PKG Iβ. Red: Residues conserved in all four domains; Yellow: Residues conserved in three of the four domains; Grey: Residues conserved between A or B domains. In PKG Iβ, R297 and T317 (indicated by arrowheads) regulate selectivity of binding for cGMP. Hence, mutation of the equivalent sites in RIα (T192R, A212T, G316R, A336T) is predicted to confer cGMP selectivity. Figure 2 Cyclic nucleotide binding of RIα constructs. Competition curves as derived from SPR solution competition experiments. Protein was pre-incubated with different concentrations of cAMP (black) or cGMP (red), respectively. Residual binding to 8-AHA-cAMP, 6-AH-cAMP and 2-AHA-cAMP surfaces was monitored. SPR binding signal without pre-incubation was set to 100% and the lowest binding signal was set to 0%. The mean of the SPR signal of two analog surfaces with standard error of the mean (SEM; error bars) was plotted against the logarithmic cyclic nucleotide concentration. Refer to Table 1. Figure 3 Cyclic nucleotide-dependent activation of RIα holoenzymes. Activation of PKA holoenzymes containing wild type or mutant RIα constructs and human catalytic subunit Cα was measured under increasing concentrations of cAMP (black) or cGMP (red), respectively. Kinase activity was normalized by setting the highest activity to 100% and basal activity (no cyclic nucleotide) to 0%. Representative plot of one protein preparation with two technical replicates. Error bars indicate SEM. Refer to Table 2. Figure 4 Restoration of PKA holoenzyme in RIα-/- cells (Prkar1a-/- MEF) by expression of RIα constructs. (A) Immunofluorescence images of non-transfected RIα-/- cells (green only) and cells transfected to express mCherry-RIα (red). Cells were labeled with an antibody directed against 21 ACS Paragon Plus Environment


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PKA-phosphorylated substrates (green) as an indicator of PKA activity. Scale bars: 20 µM. (B) Box scatter plot of relative PKA activity comparing mCherry-RIα transfected cells (red X) to non-transfected cells (blue *). Statistical analysis was performed using ANOVA; *** p < 0.001. Error bars indicate the SD of the mean of at least 33 transfected or non-transfected cells (Ctrls) for each condition (33 < n < 47). Figure 5 Measurements of PKA activation using the AKAR FRET biosensor for RIα-/- cells expressing the various RIα constructs. (A-B) Examples of RIα-/- cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t=0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 µM. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20-26, encompassing multiple independently conducted stimulations and transfections. Figure 6 The two-step model of type I PKA activation mediated by cyclic nucleotide binding. For simplicity and for space consideration, each holoenzyme is depicted as a heterodimer of one RIα and one catalytic (green) subunit. Cyclic nucleotide binding to the accessible CNB-B leads to unmasking of CNB-A. Full occupation of both CNB-A and CNB-B releases the active catalytic subunit from its RIα-bound form. While introduction of the mutations within CNB-B can modify the cyclic nucleotide binding affinities, as a whole, activation of the holoenzyme is determined by the cyclic nucleotide selectivity as determined by CNB-A. Therefore, T192R and A212T mutations introduced within CNB-A (mut CNB-A and mut CNB-A,B) changes the activation of the corresponding PKA holoenzyme from cAMP-dependent to cGMP-dependent.


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Schematics of PKA and PKG with emphasis on the cyclic nucleotide binding domains. (A) Both PKA and PKG contain dimerization/docking (D/D) domains that bind to anchoring proteins such as AKAPs and GKIPs for localization, inhibitor sites to bind to the active site cleft of the catalytic subunit, and two cyclic nucleotide binding domains, CNB-A and CNB-B. The catalytic subunits are well conserved amongst protein kinases, consisting of a N-terminal lobe for ATP binding and a C-terminal lobe for catalytic activity. While PKA exists in a tetrameric structure, PKG is a dimer, with both monomers containing a regulatory and a catalytic domain. (B) Structures of RIα complexed with cAMP and PKG Iβ complexed with cGMP are shown on the left (PDB codes 1RGS and 4Z07) and their alignments of each CNB domain on the right. In the left panel, the CNB-A domains are colored in magenta, CNB-B in teal, and PBC (phosphate binding cassette) in yellow. Cyclic GMPs are shown as stick and colored by atom type (carbon, white; nitrogen, blue; oxygen, red; and phosphorus, orange). The Cα atoms of residues important in this study are shown with red spheres. In the right panels, the CNB domains of PKG Iβ are shown in gray. (C) Structural models of CNB domain mutants. The models were generated by T192R, A212T substitutions in CNB-A, and G316R, A336T substitutions in

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CNB-B using the crystal structure of RIα as a template (PDB ID 1RGS). Key cGMP contact residues are shown as stick and hydrogen bonding interactions with cGMP are shown as dotted lines. (D) Sequence alignment of the CNB-As and CNB-Bs of RIα and PKG Iβ. Red: Residues conserved in all four domains; Yellow: Residues conserved in three of the four domains; Grey: Residues conserved between A or B domains. In PKG Iβ, R297 and T317 (indicated by arrowheads) regulate selectivity of binding for cGMP. Hence, mutation of the equivalent sites in RIα (T192R, A212T, G316R, A336T) is predicted to confer cGMP selectivity. 198x256mm (300 x 300 DPI)


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Cyclic nucleotide binding of RIα constructs. Competition curves as derived from SPR solution competition experiments. Protein was pre-incubated with different concentrations of cAMP (black) or cGMP (red), respectively. Residual binding to 8-AHA-cAMP, 6-AH-cAMP and 2-AHA-cAMP surfaces was monitored. SPR binding signal without pre-incubation was set to 100% and the lowest binding signal was set to 0%. The mean of the SPR signal of two analog surfaces with standard error of the mean (SEM; error bars) was plotted against the logarithmic cyclic nucleotide concentration. Refer to Table 1. 52x41mm (300 x 300 DPI)



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Figure 3. Cyclic nucleotide-dependent activation of RIα holoenzymes. Activation of PKA holoenzymes containing wild type or mutant RIα constructs and human catalytic subunit Cα was measured under increasing concentrations of cAMP (black) or cGMP (red), respectively. Kinase activity was normalized by setting the highest activity to 100% and basal activity (no cyclic nucleotide) to 0%. Representative plot of one protein preparation with two technical replicates. Error bars indicate SEM. Refer to Table 2. 50x38mm (300 x 300 DPI)


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Figure 4. Restoration of PKA holoenzyme in RIα-/- cells (Prkar1a-/- MEF) by expression of RIα constructs. (A) Immunofluorescence images of non-transfected RIα-/- cells (green only) and cells transfected to express mCherry-RIα (red). Cells were labeled with an antibody directed against PKA-phosphorylated substrates (green) as an indicator of PKA activity. Scale bars: 20 µM. (B) Box scatter plot of relative PKA activity comparing mCherry-RIα transfected cells (red X) to non-transfected cells (blue *). Statistical analysis was performed using ANOVA; *** p < 0.001. Error bars indicate the SD of the mean of at least 33 transfected or non-transfected cells (Ctrls) for each condition (33 < n < 47). 138x284mm (300 x 300 DPI)



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Figure 5. Measurements of PKA activation using the AKAR FRET biosensor for RIα-/- cells expressing the various RIα constructs. (A-B) Examples of RIα-/- cells expressing the indicated constructs stimulated with 8CPT-cAMP or 8-CPT-cGMP at t=0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 µM. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20-26, encompassing multiple independently conducted stimulations and transfections. 186x248mm (300 x 300 DPI)


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Figure 6. The two-step model of Type I PKA activation mediated by cyclic nucleotide binding. For simplicity and for space consideration, each holoenzyme is depicted as a heterodimer of one RIα and one catalytic (green) subunit. Cyclic nucleotide binding to the accessible CNB-B leads to unmasking of CNB-A. Full occupation of both CNB-A and CNB-B releases the active catalytic subunit from its RIα-bound form. While introduction of the mutations within CNB-B can modify the cyclic nucleotide binding affinities, as a whole, activation of the holoenzyme is determined by the cyclic nucleotide selectivity as determined by CNB-A. Therefore, T192R and A212T mutations introduced within CNB-A (mut CNB-A and mut CNB-A,B) changes the activation of the corresponding PKA holoenzyme from cAMP-dependent to cGMP-dependent. 56x47mm (300 x 300 DPI)



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Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine

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