Protein Kinase A Type I and Type II Define Distinct Intracellular Signaling Compartments Giulietta Di Benedetto, Anna Zoccarato, Valentina Lissandron, Anna Terrin, Xiang Li, Miles D. Houslay, George S. Baillie, Manuela Zaccolo Abstract—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. (Circ Res. 2008;103:836-844.) Key Words: cAMP Ⅲ compartmentalization Ⅲ compartmentation Ⅲ adrenergic stimulation Ⅲ prostaglandin Ⅲ protein kinase A rotein kinase A (PKA) is a key regulatory enzyme in the coupled to specific GPCRs to degrade cAMP selectively in Pheart that is involved in the catecholamine-mediated response to a given stimulus.8 control of excitation–contraction coupling, as well as in a Cardiac myocytes express all four types of PKA isozymes, myriad of other functions including activation of transcrip- PKA-RI␣, PKA-RII␣, PKA-RI␤, and PKA-RII␤.9 PKA iso- tion factors and control of metabolic enzymes. The second forms show different subcellular localization, with PKA-RII messenger cAMP activates PKA by binding to the regula- being mainly associated with the particulate fraction of cell tory (R) subunits, causing release of the activated catalytic lysates whereas PKA-RI has been found preferentially in the (C) subunits. cytosol.10,11 PKA isoforms also show different biochemical The fact that, following cAMP-engagement, PKA mediates properties. PKA-RI is more readily dissociated by cAMP than a plethora of cellular responses has raised the question of how PKA-RII,12,13 and the recent structure solution of holoenzyme specificity is maintained. In recent years, features of this complexes14,15 shows critical isoform-specific features that pathway that contribute to specificity have been uncovered.1 specifically regulate inhibition and cAMP-induced activation A key role is played by AKAPs (A kinase anchoring of PKA-RI and PKA-RII. Given the distinct biochemical proteins), a family of proteins that act as molecular scaffolds properties and the specific subcellular localization of PKA to anchor PKA in the vicinity of specific substrate mole- isozymes, it is not surprising that the biological role of cules,2 thus focusing PKA activity toward relevant substrates. PKA-RI and PKA-RII is nonredundant, as demonstrated by A second mechanism contributing to specificity revolves genetic and biochemical studies (reviewed elsewhere16). around the spatial control of the cAMP signal itself. Restric- However, how individual PKA isoforms serve to deliver a tion of intracellular diffusion of cAMP has been shown by specific response remains unknown. In particular, it remains using a variety of approaches,3–5 including direct imaging of to be established how spatial control of the cAMP signal and gradients of cAMP in response to activation of various G activation of individual PKA isoforms are coordinated to protein–coupled receptors (GPCRs).6 A key role in shaping perform a specific biological function. cAMP intracellular pools is played by phosphodiesterases Here, we set out to answer the question of whether (PDEs), the enzymes that hydrolyze cAMP.7 Indeed, individ- confined pools of cAMP elicited in response of specific ual PDE isoforms have been shown to be functionally extracellular stimuli selectively activate individual PKA iso- Original received February 28, 2008; revision received August 14, 2008; accepted August 19, 2008. 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 [email protected] © 2008 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.174813 836 Di Benedetto et al Compartmentalized Signaling by PKA Isoforms 837 forms. 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 compart- ments 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 tar- gets. 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. Materials and Methods Primary cultures of neonatal cardiac ventriculocytes from 1- to 3-day old rats were prepared as described.6 All the details concerning Figure 1. Targeted FRET-based cAMP sensors. Confocal generation of constructs, cells transfection, Western blotting, immu- images of CHO cells expressing either RI_epac or RII_epac nostaining and confocal imaging, FRAP experiments, FRET imaging alone (left panels) or in combination with ezrin and AKAP 79, and RT-PCR are described in the expanded Materials and Methods respectively (right images). The middle images show the local- section in the online data supplement, available at http://circres. ization of green fluorescent protein (GFP)-tagged ezrin and ahajournals.org. GFP-tagged AKAP79 in CHO cells. Scale barsϭ10 ␮m. Results RII_epac with AKAP79 results in the relocalization of the Generation of cAMP Sensors Selectively Targeted sensor at the plasma membrane, confirming that the modified to the PKA-RI and PKA-RII Compartments sensors localize within the cell where AKAPs are present. We set out to assess whether PKA-RI and PKA-RII are To verify if RI_epac and RII_epac are targeted to different selectively and independently activated by specific extracel- subcellular compartments in neonatal cardiac myocytes, the lular stimuli and generated 2 FRET-based probes that, by localization of the sensors was analyzed by confocal micros- selectively targeting to the same subcellular compartments as copy. As a localization marker, the Z-line protein Zasp fused the endogenous PKA isoforms, monitor the cAMP signals to the red fluorescent protein mRFP (zasp-RFP) was coex- generated at these sites. We took advantage of the unique pressed in combination with either RI_epac or RII_epac. As dimerization/docking domain sequences that have been illustrated in Figure 2A and 2I, RI_epac shows a tight striated shown17 to mediate anchoring of PKA-RI and PKA-RII pattern overlaying with both the Z and the M sarcomeric lines subunits to AKAPs. Thus, by fusing the dimerization/docking (see line intensity profiles at the bottom of Figure 2). In domain from either RI␣ (amino acids 1 to 64) or RII␤ (amino contrast, the distribution of RII_epac shows a very strong acids 1 to 49) to the N terminus of the soluble Epac-1 localization that corresponds to the M line and a much weaker sensor,18 we generated the sensors RI_epac and RII_epac localization overlaying the Z line (Figure 2B and 2L). Such (Figure IA in the online data supplement). For both sensors, localization is identical to the localization of overexpressed it is believed that the binding of cAMP to the cAMP-binding full-length RI and RII subunits and corresponds to the domain will result in a conformational change that causes an localization of endogenous RI and RII subunits (supplemental increase in the distance of the cyan fluorescent protein and Figure II). To assess whether the differences in localization yellow fluorescent protein moieties, with a consequent reduc- were attributable to anchoring of the sensors to endogenous tion of the FRET signal, as shown for the parent sensor.18 The AKAPs, we used the AKAP-competing peptides RIAD21 and modified sensors have been tested with respect to maximal SuperAKAP-IS.22 These peptides have been shown to com- FRET response and to dose-response behavior (supplemen- pete selectively with the binding of PKA-RI and PKA-RII to tary materials and supplemental Figure I). We found that endogenous AKAPs. In particular, the RIAD peptide displays RI_epac and RII_epac are equally sensitive to cAMP more than 1000-fold selectivity for RI over RII,21 whereas the changes. peptide SuperAKAP-IS is 10 000-fold more selective for the RII isoform relative