Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy.

BACKGROUND: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac ß-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. METHODS: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with ß-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. RESULTS: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. CONCLUSIONS: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels.

To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

Subcellular Organization of the cAMP Signaling Pathway.

The field of cAMP signaling is witnessing exciting developments with the recognition that cAMP is compartmentalized and that spatial regulation of cAMP is critical for faithful signal coding. This realization has changed our understanding of cAMP signaling from a model in which cAMP connects a receptor at the plasma membrane to an intracellular effector in a linear pathway to a model in which cAMP signals propagate within a complex network of alternative branches and the specific functional outcome strictly depends on local regulation of cAMP levels and on selective activation of a limited number of branches within the network. In this review, we cover some of the early studies and summarize more recent evidence supporting the model of compartmentalized cAMP signaling, and we discuss how this knowledge is starting to provide original mechanistic insight into cell physiology and a novel framework for the identification of disease mechanisms that potentially opens new avenues for therapeutic interventions. SIGNIFICANCE STATEMENT: cAMP mediates the intracellular response to multiple hormones and neurotransmitters. Signal fidelity and accurate coordination of a plethora of different cellular functions is achieved via organization of multiprotein signalosomes and cAMP compartmentalization in subcellular nanodomains. Defining the organization and regulation of subcellular cAMP nanocompartments is necessary if we want to understand the complex functional ramifications of pharmacological treatments that target G protein-coupled receptors and for generating a blueprint that can be used to develop precision medicine interventions.