PKG Iα Research Articles

An auto-inhibited state of protein kinase G and implications for selective activation.

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

Oxidation of PKG Iα C42 Potentiates Kinase Activation by Cyclic Nucleotides

Oxidation of PKG Iα C42 Potentiates Kinase Activation by Cyclic Nucleotides

Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

An N-terminally truncated form of cyclic GMP–dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.

Proof of Principle for a Novel Class of Antihypertensives That Target the Oxidative Activation of PKG Iα (Protein Kinase G Iα).

Arterial hypertension continues to be a major health burden. Development of new antihypertensive drugs that engage vasodilatory mechanisms not harnessed by available therapies offer therapeutic potential. Oxidants induce an interprotein disulfide in PKG Iα (protein kinase G Iα) at C42, which is associated with its targeting and activation, resulting in vasodilation and blood pressure lowering. Consequently, we developed an assay and screened for electrophilic drugs that activate PKG Iα by selectively targeting C42, as such compounds have potential as novel antihypertensives with a mechanism of action that differs from current therapies. In this way, a drug that we termed G1 was identified, which targets C42 of PKG Iα to induce vasodilation of isolated resistance blood vessels and blood pressure lowering in a mouse model of angiotensin II-induced hypertension. In contrast, these antihypertensive effects were deficient in angiotensin II-induced hypertensive C42S PKG Iα knockin mice. These transgenic mice were engineered to have the reactive cysteinyl thiol replaced with a hydroxyl so that it cannot react with endogenous vasodilatory oxidants or electrophiles such as drug G1. These studies, therefore, provide validation of PKG Iα C42 as the target of G1, as well as proof-of-principle for a new class of antihypertensive drugs that have potential for further development for clinical use in humans.

Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity

BackgroundProtein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress. Methods and resultsBovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)−1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5′-triphosphate-cyclohydrolase 2 to prevent BH4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH4 from BH2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or KI mice were then subjected to a diet-induced obesity protocol by feeding them with a high fat Western-type diet (RM 60% AFE) for 27 weeks, which increased body mass, adiposity, plasma leptin, resistin and glucagon levels comparably in each genotype. Obesity-induced hypertension, assessed by radiotelemetry, was mild and transient in the WT, while the basally hypertensive KI mice were resistant to further increases in blood pressure following high fat feeding. Although the obesogenic diet caused mild cardiac dysfunction in the WT but not the KI mice, gross changes in myocardial structure monitored by echocardiography were not apparent in either genotype. The level of cyclic guanosine monophosphate (cGMP) was decreased in the aortae of WT and KI mice following high fat feeding. PKG Iα oxidation was not evident in the hearts of WT mice fed a high fat diet. ConclusionsDespite robust evidence for PKG Iα oxidation during NOS uncoupling in cell models, it is unlikely that PKG Iα oxidation occurs to a significant extent in vivo during diet-induced obesity and so is unlikely to mediate the associated cardiovascular dysfunction.

Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation

Phosphodiesterase 5 (PDE5) inhibitors limit myocardial injury caused by stresses, including doxorubicin chemotherapy. cGMP binding to PKG Iα attenuates oxidant-induced disulfide formation. Because PDE5 inhibition elevates cGMP and protects from doxorubicin-induced injury, we reasoned that this may be because it limits PKG Iα disulfide formation. To investigate the role of PKG Iα disulfide dimerization in the development of apoptosis, doxorubicin-induced cardiomyopathy was compared in male wild type (WT) or disulfide-resistant C42S PKG Iα knock-in (KI) mice. Echocardiography showed that doxorubicin treatment caused loss of myocardial tissue and depressed left ventricular function in WT mice. Doxorubicin also reduced pro-survival signaling and increased apoptosis in WT hearts. In contrast, KI mice were markedly resistant to the dysfunction induced by doxorubicin in WTs. In follow-on experiments the influence of the PDE5 inhibitor tadalafil on the development of doxorubicin-induced cardiomyopathy in WT and KI mice was investigated. In WT mice, co-administration of tadalafil with doxorubicin reduced PKG Iα oxidation caused by doxorubicin and also protected against cardiac injury and loss of function. KI mice were again innately resistant to doxorubicin-induced cardiotoxicity, and therefore tadalafil afforded no additional protection. Doxorubicin decreased phosphorylation of RhoA (Ser-188), stimulating its GTPase activity to activate Rho-associated protein kinase (ROCK) in WTs. These pro-apoptotic events were absent in KI mice and were attenuated in WTs co-administered tadalafil. PKG Iα disulfide formation triggers cardiac injury, and this initiation of maladaptive signaling can be blocked by pharmacological therapies that elevate cGMP, which binds kinase to limit its oxidation.

Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα

PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.

Structures of cGMP-Dependent Protein Kinase (PKG) Iα Leucine Zippers Reveal an Interchain Disulfide Bond Important for Dimer Stability

cGMP-dependent protein kinase (PKG) Iα is a central regulator of smooth muscle tone and vasorelaxation. The N-terminal leucine zipper (LZ) domain dimerizes and targets PKG Iα by interacting with G-kinase-anchoring proteins. The PKG Iα LZ contains C42 that is known to form a disulfide bond upon oxidation and to activate PKG Iα. To understand the molecular details of the PKG Iα LZ and C42-C42' disulfide bond, we determined crystal structures of the PKG Iα wild-type (WT) LZ and C42L LZ. Our data demonstrate that the C42-C42' disulfide bond dramatically stabilizes PKG Iα and that the C42L mutant mimics the oxidized WT LZ structurally.

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40-83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.

PSS167 - Cys42Ser PKG Iα Mutant Mice Are Protected from Doxorubicin-Induced Cardiomyopathy

PSS167 - Cys42Ser PKG Iα Mutant Mice Are Protected from Doxorubicin-Induced Cardiomyopathy

Sulfhydryl-dependent Dimerization and cGMP-mediated Vasodilatation

Sulfhydryl-dependent formation of interprotein disulfide bonds in response to physiological oxidative stimuli is emerging as an important mechanism in the regulation of various biological activities. Soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) are key enzymes for actions caused by cGMP-elevating agents, including nitric oxide (NO). Both sGC and PKG are dimers. The dimerization of sGC is obligatory for its activity, whereas the dimerization of PKG improving its signaling efficacy. sGC dimerization is decreased by endogenous and exogenous thiol reductants, associated with reduced cGMP elevation and attenuated vasodilatation to NO. The dimerization of PKG Iα is increased by oxidative stress, coincident with improved PKG signaling and augmented vasodilatation to NO. In coronary arteries, the dimerizations and activities of sGC and PKG are increased by hypoxia, accompanied by enhanced relaxation induced by NO. In contrast, the dimerizations and activities of these enzymes and NO-induced relaxation of pulmonary arteries are reduced by hypoxia. These opposite effects may result from divergent changes in the redox status of cytoplasmic reduced nicotinamide adenine dinucleotide phosphate between coronary and pulmonary arteries in response to hypoxia.

Protein kinase G oxidation is a major cause of injury during sepsis

Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output. These responses are typical features of sepsis and their combined effect is a lowering of blood pressure. This hypotension, a hallmark of sepsis, resulted in underperfusion of end organs, resulting in their damage. A central role for PKG Iα oxidative activation in injury is supported by oxidation-resistant Cys42Ser PKG Iα knock-in mice being markedly protected from these clinical indices of injury during sepsis. We conclude that oxidative activation of PKG Iα is a key mediator of hypotension and consequential organ injury during sepsis.

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including "ecstasy" (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N(6)-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-(33)P]ATP but not from similarly labeled N(6)-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Atorvastatin inhibits collar-induced intimal thickening of rat carotid artery: Effect on C-type natriuretic peptide expression

The present study was performed to elucidate the mechanism underlying the anti-atherogenic action of atorvastatin (ATV). We investigated the effect of ATV on intimal thickening of the rat carotid artery induced by collar placement and further examined the effect of ATV on the expression of C-type natriuretic peptide (CNP), cGMP-dependent protein kinase (PKG) Iα and PKG Iβ in carotid arteries. The expression of CNP was examined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time RT-PCR. Western blotting was used to determine the expression of PKG Iα and PKG Iβ. After 14 days, the collar placement induced a marked neointima formation and a reduction in CNP and PKG Iα expression. These effects were significantly reversed by ATV treatment. However, no obvious changes in PKG Iβ expression were observed throughout the study. In conclusion, the present data suggest that elevation of CNP represents an additional mechanism by which ATV treatment may prevent the development and progression of atherosclerosis.

Crystal Structure of cGMP-Dependent Protein Kinase Reveals Novel Site of Interchain Communication

The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory, and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here, we report the 2.5Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full-length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.

The amino terminus of cGMP-dependent protein kinase Iβ increases the dynamics of the protein's cGMP-binding pockets

The type I cGMP-dependent protein kinases play critical roles in regulating vascular tone, platelet activation and synaptic plasticity. PKG Iα and PKG Iβ differ in their first ∼100 amino acids giving each isoform unique dimerization and autoinhibitory domains with identical cGMP-binding pockets and catalytic domains. The N-terminal leucine zipper and autoinhibitory domains have been shown to mediate isoform specific affinity for cGMP. PKG Iα has a >10-fold higher affinity for cGMP than PKG Iβ, and PKG Iβ that is missing its leucine zipper has a 3-fold decreased affinity for cGMP. The exact mechanism through which the N-terminus of PKG alters cGMP-affinity is unknown. In the present study, we have used deuterium exchange mass spectrometry to study how PKG Iβ’s N-terminus affects the conformation and dynamics of its cGMP-binding pockets. We found that the N-terminus increases the rate of deuterium exchange throughout the cGMP-binding domain. Our results suggest that the N-terminus shifts the conformational dynamics of the binding pockets, leading to an “open” conformation that has an increased affinity for cGMP.

CGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity

Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP 3) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG Iβ, but not PKG Iα, and in smooth muscle cells, IRAG and PKG Iβ are in a complex with the IP 3 receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG Iβ in baby hamster kidney cells prevented cGMP-induced PKG Iβ translocation to the nucleus, and decreased cGMP/PKG Iβ transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG Iβ/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG Iβ, as nuclear translocation and reporter gene activation by PKG Iα was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG Iβ transcriptional activity, indicating that IRAG's effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types.