Anna Terrin

Fluorescence Resonance Energy Transfer–Based Analysis of cAMP Dynamics in Live Neonatal Rat Cardiac Myocytes Reveals Distinct Functions of Compartmentalized Phosphodiesterases

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a β-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

Compartmentalized Phosphodiesterase-2 Activity Blunts β-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway

&bgr;-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving &bgr;3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by &bgr;-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the &bgr;-adrenergic pathway. In this, activation of &bgr;3-adrenergic receptors counteracts cAMP generation obtained via stimulation of &bgr;1/&bgr;2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Protein kinase A type I and type II define distinct intracellular signaling compartments.

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.

PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases.

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

The Role of Type 4 Phosphodiesterases in Generating Microdomains of cAMP: Large Scale Stochastic Simulations

Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

The Mitochondrial Calcium Uniporter (MCU): Molecular Identity and Physiological Roles

The direct measurement of mitochondrial [Ca2+] with highly specific probes demonstrated that major swings in organellar [Ca2+] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca2+ uniporter, the channel allowing rapid Ca2+ accumulation across the inner mitochondrial membrane.

Restricted diffusion of a freely diffusible second messenger: mechanisms underlying compartmentalized cAMP signalling.

It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed.

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals through PKA and PDE4D3 interaction with the A kinase anchoring protein AKAP9.

A Phosphodiesterase 3B-based Signaling Complex Integrates Exchange Protein Activated by cAMP 1 and Phosphatidylinositol 3-Kinase Signals in Human Arterial Endothelial Cells

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based “signalosome” that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.