Published online before print August 28, 2008, doi: 10.1161/CIRCRESAHA.108.174813
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© 2008 American Heart Association, Inc.
Cellular Biology |
Protein Kinase A Type I and Type II Define Distinct Intracellular Signaling Compartments
From the Dulbecco Telethon Institute (G.D.B., A.Z., V.L., M.Z.), Venetian Institute of Molecular Medicine, Padova, Italy; and Neuroscience and Molecular Pharmacology (A.T., X.L., M.D.H., G.S.B., M.Z.), Faculty of Biomedical & Life Sciences, University of Glasgow, Scotland, United Kingdom.
Correspondence to Dr Manuela Zaccolo, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University Avenue, G12 8QQ, Glasgow, UK. E-mail m.zaccolo{at}bio.gla.ac.uk
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Abstract |
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Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein–coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.
Key Words: cAMP • compartmentalization • compartmentation • adrenergic stimulation • prostaglandin • protein kinase A
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein kinase A (PKA) is a key regulatory enzyme in the heart that is involved in the catecholamine-mediated control of excitation–contraction coupling, as well as in a myriad of other functions including activation of transcription factors and control of metabolic enzymes. The second messenger cAMP activates PKA by binding to the regulatory (R) subunits, causing release of the activated catalytic (C) subunits.
The fact that, following cAMP-engagement, PKA mediates a plethora of cellular responses has raised the question of how specificity is maintained. In recent years, features of this pathway that contribute to specificity have been uncovered.1 A key role is played by AKAPs (A kinase anchoring proteins), a family of proteins that act as molecular scaffolds to anchor PKA in the vicinity of specific substrate molecules,2 thus focusing PKA activity toward relevant substrates.
A second mechanism contributing to specificity revolves around the spatial control of the cAMP signal itself. Restriction of intracellular diffusion of cAMP has been shown by using a variety of approaches,3–5 including direct imaging of gradients of cAMP in response to activation of various G protein–coupled receptors (GPCRs).6 A key role in shaping cAMP intracellular pools is played by phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP.7 Indeed, individual PDE isoforms have been shown to be functionally coupled to specific GPCRs to degrade cAMP selectively in response to a given stimulus.8
Cardiac myocytes express all four types of PKA isozymes, PKA-RI
, PKA-RII, PKA-RIβ, and PKA-RIIβ.9 PKA isoforms show different subcellular localization, with PKA-RII being mainly associated with the particulate fraction of cell lysates whereas PKA-RI has been found preferentially in the cytosol.10,11 PKA isoforms also show different biochemical properties. PKA-RI is more readily dissociated by cAMP than PKA-RII,12,13 and the recent structure solution of holoenzyme complexes14,15 shows critical isoform-specific features that specifically regulate inhibition and cAMP-induced activation of PKA-RI and PKA-RII. Given the distinct biochemical properties and the specific subcellular localization of PKA isozymes, it is not surprising that the biological role of PKA-RI and PKA-RII is nonredundant, as demonstrated by genetic and biochemical studies (reviewed elsewhere16). However, how individual PKA isoforms serve to deliver a specific response remains unknown. In particular, it remains to be established how spatial control of the cAMP signal and activation of individual PKA isoforms are coordinated to perform a specific biological function.
Here, we set out to answer the question of whether confined pools of cAMP elicited in response of specific extracellular stimuli selectively activate individual PKA isoforms. By using FRET- and FRAP-based imaging approaches we show that, in cardiomyocytes, PKA-RI and PKA-RII, by anchoring to endogenous AKAPs, define distinct compartments within which cAMP is specifically controlled by different subsets of PDEs. In addition, we demonstrate that cAMP levels rise selectively in the PKA-RI and PKA-RII compartments in a stimulus-specific manner, leading to the phosphorylation of unique subsets of downstream PKA targets. The generation of distinct pools of cAMP within cells that allows for the selective activation of individual PKA isoforms is instrumental for the cell to modulate specific physiological functions and points to means for developing strategies for selective pharmacological intervention.
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Materials and Methods |
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Primary cultures of neonatal cardiac ventriculocytes from 1- to 3-day old rats were prepared as described.6 All the details concerning generation of constructs, cells transfection, Western blotting, immunostaining and confocal imaging, FRAP experiments, FRET imaging and RT-PCR are described in the expanded Materials and Methods section in the online data supplement, available at http://circres. ahajournals.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of cAMP Sensors Selectively Targeted to the PKA-RI and PKA-RII Compartments
We set out to assess whether PKA-RI and PKA-RII are selectively and independently activated by specific extracellular stimuli and generated 2 FRET-based probes that, by selectively targeting to the same subcellular compartments as the endogenous PKA isoforms, monitor the cAMP signals generated at these sites. We took advantage of the unique dimerization/docking domain sequences that have been shown17 to mediate anchoring of PKA-RI and PKA-RII subunits to AKAPs. Thus, by fusing the dimerization/docking domain from either RI
(amino acids 1 to 64) or RIIβ (amino acids 1 to 49) to the N terminus of the soluble Epac-1 sensor,18 we generated the sensors RI_epac and RII_epac (Figure IA in the online data supplement). For both sensors, it is believed that the binding of cAMP to the cAMP-binding domain will result in a conformational change that causes an increase in the distance of the cyan fluorescent protein and yellow fluorescent protein moieties, with a consequent reduction of the FRET signal, as shown for the parent sensor.18 The modified sensors have been tested with respect to maximal FRET response and to dose-response behavior (supplementary materials and supplemental Figure I). We found that RI_epac and RII_epac are equally sensitive to cAMP changes.
RI_epac and RII_epac Show a Different and AKAP-Mediated Localization in Cardiac Myocytes
To assess the ability of the modified sensors to effectively bind to AKAPs, we coexpressed RI_epac with ezrin and RII_epac with AKAP79. Ezrin is a dual-specificity AKAP localized at the cortical cytoskeleton and at microvilli,19 whereas AKAP79 localizes at the plasma membrane (Figure 1).20 Coexpression in CHO cells of RI_epac with ezrin results in the relocalization of the sensor to the sub–plasma membrane region and to microvilli, whereas coexpression of RII_epac with AKAP79 results in the relocalization of the sensor at the plasma membrane, confirming that the modified sensors localize within the cell where AKAPs are present.
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To verify if RI_epac and RII_epac are targeted to different subcellular compartments in neonatal cardiac myocytes, the localization of the sensors was analyzed by confocal microscopy. As a localization marker, the Z-line protein Zasp fused to the red fluorescent protein mRFP (zasp-RFP) was coexpressed in combination with either RI_epac or RII_epac. As illustrated in Figure 2A and 2I, RI_epac shows a tight striated pattern overlaying with both the Z and the M sarcomeric lines (see line intensity profiles at the bottom of Figure 2). In contrast, the distribution of RII_epac shows a very strong localization that corresponds to the M line and a much weaker localization overlaying the Z line (Figure 2B and 2L). Such localization is identical to the localization of overexpressed full-length RI and RII subunits and corresponds to the localization of endogenous RI and RII subunits (supplemental Figure II). To assess whether the differences in localization were attributable to anchoring of the sensors to endogenous AKAPs, we used the AKAP-competing peptides RIAD21 and SuperAKAP-IS.22 These peptides have been shown to compete selectively with the binding of PKA-RI and PKA-RII to endogenous AKAPs. In particular, the RIAD peptide displays more than 1000-fold selectivity for RI over RII,21 whereas the peptide SuperAKAP-IS is 10 000-fold more selective for the RII isoform relative to RI.22 Challenge of cells expressing RI_epac with RIAD and of cells expressing RII_epac with SuperAKAP-IS completely abolished the striated pattern of localization of the sensors (Figure 2C and 2D), whereas RIAD and SuperAKAP-IS did not have any effect on the localization pattern of, respectively, RII_epac and RI_epac (not shown).
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The accepted paradigm for the PKA signaling pathway is that type II PKA is associated with particulate subcellular fractions via binding to AKAPs, whereas type I PKA is primarily cytoplasmic.11 Our confocal imaging studies suggest, however, that RI is also anchored in neonatal cardiac myocytes. To assess the extent of anchoring of the RI_epac and RII_epac sensors, we conducted fluorescence recovery after photobleaching (FRAP) experiments. As shown in Figure 3A, following fluorescence bleaching in cells expressing either the untagged, cytosolic cAMP sensor Epac-1 or 1 of the targeted sensors, we found that fluorescence recovery occurred with a t1/2 of 5.7±0.4 seconds (n=12) for Epac1, 14.3±1.9 seconds (n=10) for RI_epac, and 21.6±1.7 seconds (n=10) for RII_epac. Fluorescence recovery was almost complete for the parent Epac-1 probe (fractional recovery of 94.2±1.2%). However, this was reduced to 73.1±3.4% for RI_epac and to 45.4±5.0% for RII_epac. These results confirm that although the Epac-1 parent probe is free to diffuse in the cytosol, the diffusion of both RI_epac and RII_epac is substantially constrained. However, despite the mobility of both the RI_epac and RII_epac being restricted, we noted that RI_epac was 1.6 times as mobile as RII_epac.
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To assess the extent to which the reduced mobility of RI_epac and RII_epac was attributable to anchoring to endogenous AKAPs, we repeated the FRAP experiments in cardiomyocytes expressing each of the targeted sensor in combination with specific competing peptides. Figure 3B shows a significant increase in the fractional recovery both for cells expressing RI_epac in combination with RIAD (85.3±2.3% [n=10]) and for cells expressing RII_epac in combination with SuperAKAP-IS (80.1±2.5% [n=11]). Accordingly, we found a decrease in the recovery times with a t1/2 of 8.4±0.8 seconds (n=10) in cells expressing RI_epac+RIAD and of 8.9±0.9 seconds (n=11) in cells expressing RII_epac+SuperAKAP-IS. These results demonstrate that RI_epac and RII_epac specifically localize to different intracellular compartments primarily by binding to specific endogenous AKAPs.
cAMP Levels Are Specifically Regulated in the PKA-RI and PKA-RII Compartments
In a first set of experiments, we wanted to assess whether the PKA-RI and PKA-RII compartments have equal access to cAMP. Cardiac myocytes expressing either RI_epac or RII_epac were challenged with 5 µmol/L forskolin. As shown in Figure 4A and 4F, the 2 sensors detected a comparable rise in [cAMP] with 
R/R0=2.1±0.4% (n=7) for RI_epac and R/R0=2.9±0.4% (n=14) for RII_epac (P=0.24), indicating that the compartments hosting PKA-RI and PKA-RII are equally associated to adenylyl cyclases and have potentially access to comparable cAMP levels.
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Next, we wanted to assess the role of PDEs in the control of cAMP levels in both PKA compartments. When myocytes were challenged with isobutylmethylxanthine (IBMX) (100 µmol/L), no significant difference was detected in the level of cAMP in the 2 compartments (R/R0=4.5±0.3%; n=20 for RI_epac and R/R0=4.9±0.4%; n=15 for RII_epac; P=0.45) (Figure 4B and 4F), suggesting that the basal level of cAMP is under comparable levels of PDE control in both PKA-RI and PKA-RII compartments in these cells.
To determine whether specific subsets of PDEs control the cAMP signal in the 2 PKA compartments, cardiac myocytes expressing either RI_epac or RII_epac were treated with selective PDE inhibitors. As shown in Figure 4C and 4F, PDE3 inhibition assessed using the selective inhibitor cilostamide (10 µmol/L) generated a small and similar cAMP rise in both PKA compartments (R/R0=0.8±0.2%; n=9 for RI_epac and R/R0=0.6±0.2%; n=11 for RII_epac; P=0.58). PDE2 inhibition assessed using the selective inhibitor, EHNA (10 µmol/L) resulted in a R/R0=2.2±0.8% (n=15) when detected by RI_epac and in a R/R0=0.3±0.2% (n=12) when detected by RII_epac (P=0.04), indicating that, in basal conditions, PDE2 activity is prominent in the PKA-RI compartment but very low in the RII_epac compartment (Figure 4D and 4F). PDE4 inhibition, assessed using the selective inhibitor rolipram (10 µmol/L), generated a R/R0=0.7±0.2% (n=13) when detected by RI_epac and a R/R0=2.6±0.7% (n=16) when detected with RII_epac (P=0.02) (Figure 4E and 4F), indicating that, contrary to PDE2, PDE4 exerts its activity mainly in the PKA-RII domain and to a much lower extent in the PKA-RI compartment. These results show that cAMP levels are differently regulated in the PKA-RI and PKA-RII compartments and point to a specific association of individual PKA isoforms with selected subsets of PDEs in these cells.
Individual GPCRs Generate a cAMP Signal Selectively in the PKA-RI or PKA-RII Compartments
We next asked whether the PKA-RI and PKA-RII compartments may be coupled to specific GPCRs such that individual PKA isoforms respond selectively to cAMP signals elicited by different agonists. As shown in Figure 5A, the application of isoproterenol (10 nmol/L), a specific activator of β-adrenoreceptors, generated a rise in [cAMP] more pronounced in the PKA II domain as compared to the PKA I domain (R/R0=2.5±0.5% [n=13] for RI_epac and R/R0=4.5±0.7% [n=9] for RII_epac; P=0.007). Addition of IBMX (100 µmol/L) abolishes such a difference. This result is in agreement with our previous studies showing that β-adrenergic stimulation generates a restricted pool of cAMP that activates selectively AKAP-anchored PKA-RII.6 By contrast, challenging the myocytes with GLP-1 (100 nmol/L) resulted in a larger [cAMP] increase in the PKA-RI compartment as compared to the PKA-RII compartment (R/R0=3.7±0.8%, n=15 for RI_epac and R/R0=1.0±0.4%, n=12, for RII_epac; P=0.008) (Figure 5B). Similarly, 300 nmol/L glucagon (Figure 5C) and 1 µmol/L prostaglandin E1 (PGE1) (Figure 5D) generated a higher [cAMP] increase in the PKA-RI associated compartment than in the PKA-RII compartment, showing R/R0=1.3±0.4% (n=7) for RI_epac and R/R0=0.2±0.1% (n=6) for RII_epac (P=0.017) in the case of glucagon and R/R0=1.4±0.2% (n=35) for RI_epac and R/R0=0.6±0.2% (n=30) for RII_epac (P=0.01) in the case of PGE1. In the presence of the specific competing peptide RIAD, the cAMP signal generated by the application of isoproterenol (10 nmol/L) was now clearly detected by RI_epac (R/R0=5.0±1.0% [n=9]; P=0.008 versus RI_epac alone; Figure 5F). Coexpression of SuperAKAP-IS and RII_epac did not affect RII_epac ability to detect the isoproterenol-induced cAMP signal (Figure 5F). Similarly, when RII_epac was coexpressed with the selective competing peptide SuperAKAP-IS, the amplitude of the cAMP signal generated by application of 1 µmol/L PGE1 was now clearly detected by RII_epac (R/R0=2.8±0.7% [n=17]; P=0.0001 versus RII_epac alone; Figure 5G), whereas coexpression of RIAD and RI_epac had no effect on the amplitude of the signal detected on PGE1 application (Figure 5G). In addition, we found that treatment with RIAD significantly reduced the velocity of the FRET change of RI_epac to PGE1 (supplemental Figure III).
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Taken together, these results demonstrate that individual GPCR agonists generate spatially restricted pools of cAMP that selectively activate individual AKAP-anchored PKA isoforms. Removal of PKA isoforms from their anchoring sites allows them to diffuse and to be activated by pools of cAMP that would not normally affect them.
Compartmentalized PKA Isozymes Phosphorylate Selected Subsets of Downstream Targets
We next wanted to assess whether the selectivity of the cAMP signals generated in the PKA-RI and PKA-RII compartments on stimulation of individual GPCRs has a functional relevance and results in a specific pattern of PKA-mediated phosphorylation of downstream targets. To this aim, we studied the phosphorylation level of several PKA targets after stimulation with either isoproterenol or PGE1. The phosphorylation level of phospholamban (PLB), troponin (Tn)I, and the β adrenergic receptor type 2 (β2AR) was markedly increased (Figure 6A) on stimulation with isoproterenol (1 nmol/L) but not on stimulation with PGE1 (10 µmol/L). Stimulation of myocytes with isoproterenol in the presence of the PKA inhibitor KT5720 (2 µmol/L) completely abolished the increased phosphorylation level of both PLB and TnI (Figure 6B), confirming the involvement of PKA. Unexpectedly, PGE1-stimulated cells showed a reduction in the phosphorylation level of PLB and TnI as compared to untreated cells. Further analysis indicated that the reduced phosphorylation of these targets was dependent on the activity of phosphatases (supplemental Figure IV).
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Based on these results we can conclude that individual agonists, via activation of specific PKA isoforms, affect distinct subsets of downstream targets.
A Gi-Mediated Mechanism Contributes to Control the cAMP Signal, Leading to the Phosphorylation of PLB and TnI
Four different receptors for PGE have been described (EP1 to EP4) with EP2 and EP4 being coupled to Gs and EP3 being coupled to Gi.23 We asked whether the reduction in the phosphorylation level of PBL and TnI observed on PGE1 stimulation may be attributable to activation of an EP3 receptor and subsequent Gi-mediated inhibition of cyclase activity. RT-PCR analysis of mRNA extracted from neonatal rat cardiomyocytes confirmed that these cells express high levels of message for the Gi-coupled EP3 receptor, as well as message for the Gs-coupled EP2 and EP4 receptors (Figure 7A).
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To evaluate the occurrence and the relevance of Gi activation on the local control of the cAMP signal generated by PGE1, we performed imaging experiments applying PGE1 on cardiomyocytes that had been pretreated with the Gi-inhibitor pertussis toxin (PTX). As shown in Figure 7B, application of 1 µmol/L PGE1 following 2 to 4 hours pretreatment with 2 µg/mL PTX generated a comparable cAMP increase in the PKA-RI and PKA-RII compartments. In particular, PTX pretreatment results in a much-increased cAMP signal in the PKA-RII compartment (R/R0=1.7±0.4% [n=11] versus 0.6±0.2% [n=30] in the absence of PTX; P=0.006). Indeed, this equals the amplitude of the signal generated in the PKA-RI compartment (R/R0=1.4±0.2%; P=0.54; see Figure 7B). No significant effect of PTX pretreatment was observed on the amplitude of the cAMP signal observed in the PKA-RI compartment (R/R0=2.1±0.5% [n=8] as compared to R/R0=1.4%±0.2% in control cells; P=0.13).
To assess the functional relevance of such a Gi-mediated control of the cAMP signal, we measured the level of phosphorylation of PLB and TnI in cardiomyocytes pretreated with PTX for 1, 2, or 4 hours before the application of PGE1. Figure 7C shows that, on inhibition of Gi, PGE1 stimulation induce a significant increase in the phosphorylation level of both these PKA-RII–specific targets (compare Figures 6 and 7
C). Thus, a Gi-mediated mechanism appears to contribute significantly to the control of the cAMP signal generated by PGE1 stimulation in the PKA-RII domain.
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Discussion |
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The organization of the signaling machinery in discrete compartments is increasingly recognized as a critical feature for the specificity of the cAMP/PKA system in cardiomyocytes.24 Here, we used real-time imaging of cAMP to study how the diversity of PKA isoforms may contribute to the transduction of specific downstream signals. We did this by targeting FRET-based reporters of cAMP concentration at intracellular sites where endogenous PKA-RI and PKA-RII isoforms localize. We demonstrate that PKA-RI and PKA-RII reside in physically segregated and distinct compartments within which the level of cAMP appears to be selectively regulated by different subsets of PDEs, although the exact contribution of individual PDEs would require simultaneous inhibition of combinations of different isoforms. We show that the cAMP signal is specifically generated either in the PKA-RI or in the PKA-RII compartment depending on the GPCR agonist applied and that cAMP does not diffuse from one compartment to the other so as to cross-activate PKA isozymes, allowing fidelity of the response (Figure 8). One functional consequence of such compartmentalization is that isoproterenol stimulation leads to the specific phosphorylation of PLB, TnI, and β2AR, whereas PGE1 stimulation does not affect these substrates, demonstrating that individual PKA isoforms are coupled with defined subsets of targets and that PKA isoforms activity is not promiscuous.
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Our findings are consistent with previous work25–27 showing that, in cardiac myocytes, there is a clear dichotomy between the effects of PGE1 and isoproterenol on a variety of cAMP-dependent events. These studies demonstrated that isoproterenol generates an increase in contractile force25 enhances ventricular pressure development and induces phosphorylation of target proteins such as phosphorylase kinase and TnI,26 whereas PGE1, although elevating cAMP to comparable levels and inducing activation of PKA similar to isoproterenol, does not show any effect on contractility or on the phosphorylation of these target proteins.
The general notion concerning subcellular localization of PKA isoforms in cardiac myocytes is that PKA-RII holoenzyme is localized whereas PKA-RI is mainly cytosolic. Biochemical studies on cell homogenates showed that PKA-RII subunits are bound to particulate material in heart cells, whereas RI subunits are largely soluble.10,11 Interestingly, the selective effects of β-adrenergic stimulation on heart function has been shown to correlate with activation of a membrane-bound fraction of PKA, whereas PGE1 stimulation increases the activity of a soluble pool of PKA.26 Our data now provide a mechanism for these observation by showing that catecholamines generate restricted pools of cAMP that selectively engage PKA-RII isoforms. However, the notion of a PKA-RI migrating freely in the cytosol, as suggested on the basis of previous biochemical studies, is difficult to reconcile with any selectivity in the activation of PKA isozymes, particularly considering that PKA-RI is more readily activated by cAMP as compared to PKA-RII.13 In tissues other than the heart, dual-specificity AKAPs capable of binding PKA-RI and -RII have been described,28 and AKAP-mediated localization of PKA-RI to the neuromuscular junction29 or to interphase microtubules and specific regions of the mitotic spindle has been shown.30 The discovery in Caenorhabditis elegans of a specific RI-binding AKAP that does not interact with RII subunits31 suggests the potential for nonredundant PKA-RI localization and function and a specific role for localized PKA-RI in modulating T-cell receptor signaling has been demonstrated.32 Thus, by performing FRAP experiments on intact cells, here we find that neonatal cardiac myocytes express a considerable amount of PKA-RI anchor sites, as shown by the slow fluorescence recovery time and the large immobile fraction detected in cells expressing the RI_epac sensor. This suggests that a large fraction of the PKA-RI isozyme may in fact be anchored in the heart in vivo. The fact that PKA-RI is found mainly in the supernatant of heart cell homogenates may reflect relatively low affinity interactions between PKA-RI and the protein binding partners that are lost during the homogenization and washing steps. Notwithstanding this, the nature of the PKA-RI anchoring proteins in cardiac myocytes remains to be established.
Catecholamine-mediated sympathetic control of cardiac stimulation is a major compensatory mechanism that maintains or augments systolic and diastolic ventricular function during physiological stress or pathological conditions. In particular, catecholamines selectively improve diastolic function by reducing myofilament calcium sensitivity through phosphorylation of proteins such as TnI and accelerate sequestration of calcium into the sarcoplasmic reticulum through phosphorylation of PLB and release of its inhibitory effect on the SERCA pump.33 Enhanced and sustained cardiac adrenergic drive, however, is known to be deleterious and to contribute, in part, to development and progression of pathological states such as heart failure.34 Our results indicate that key proteins involved in the catecholamine-mediated regulation of cardiac contractility are under the control of a restricted pool of cAMP that selectively activates a subset of PKA-RII isozymes, thus leading to a coordinated and specific response. In addition, we show that a supplementary element contributing to specificity is provided by a Gi-mediated mechanism that contributes to keep the PKA-RII compartment clear of cAMP unless the appropriate catecholaminergic stimulus impinges on the cell. Such a mechanism may indeed serve to protect the heart from excessive adrenergic stimulation in pathological conditions. Interestingly, PGE1 stimulation is known to protect myocardial tissue from injury following ischemia and reperfusion,35 an effect that has been suggested to depend on PGE-mediated stimulation of EP3 receptors in cardiac myocytes36 and the consequent Gi-mediated inhibition of cAMP synthesis.37
In summary, the present work provides original insight into the mechanisms that underpin cAMP/PKA specificity of response by demonstrating that, in cardiac myocytes, both PKA-RI and PKA-RII isoforms subsets anchor to subcellular sites via binding to endogenous AKAPs. This defines exclusive signaling domains within which the cAMP signal is uniquely generated via activation of specific GPCR and their associated G proteins and is uniquely modulated by the activity of different subsets of PDEs, resulting in stimulus-specific phosphorylation of downstream protein targets. These results provide a mechanism for the different function of PKA-RI and PKA-RII subsets and provide a functional rationale for the design of isoform-specific activators and inhibitors of PKA.
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Acknowledgments |
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We thank Martin Lohse for providing Epac-1, John Scott and Kjetil Tasken for the constructs for RIAD and for SuperAKAP-IS, Kjetil Tasken for ezrin and ezrin-GFP, Matteo Vatta for wt-Cypher/ZASP-GFP, and Marc Dell’Acqua for AKAP-79 and AKAP-79-GFP.
Sources of Funding
This work was supported by Telethon Italy grant GGP05113; Human Frontier Science Program Organization grant RGP0001/2005-C (to M.Z.); Fondation Leducq grant O6 CVD 02 (to M.Z.); and the European Commission grant LSHB-CT-2006-037189 (to M.Z., G.S.B., and M.D.H.); and Medical Research Council grant G0600765 (to M.D.H. and G.S.B.).
Disclosures
None.
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Footnotes |
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Original received February 28, 2008; revision received August 14, 2008; accepted August 19, 2008.
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References |
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