Regulation of Nuclear PKA revealed by spatiotemporal manipulation of cAMP

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Nature Chemical Biology (Impact Factor: 13). 02/2012; 8(4):375-82. DOI: 10.1038/nchembio.799
Source: PubMed


Understanding how specific cyclic AMP (cAMP) signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncovered the activation of a resident pool of protein kinase A (PKA) holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases and A-kinase anchoring proteins (AKAPs) are critical in shaping nuclear PKA responses. Collectively, our data suggest a new model in which AKAP-localized phosphodiesterases tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

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Available from: Jeffrey J Saucerman, Sep 30, 2015
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    • "ocalization signals , or membrane anchoring motifs ( farnesylation , palmitoylation , or polybasic sequences ) , several sensors were successfully routed to desired compartments , including plasma membrane , cytoplasm , mitochondria , and nucleus ( DiPilato et al . , 2004 ; Ponsioen et al . , 2004 ; Dyachok et al . , 2006 ; Terrin et al . , 2006 ; Sample et al . , 2012 ) . This very ability to provide outstanding spatial resolution , unattainable with other biosensors , combined with quick response and reversible nature of conformational changes induced upon cAMP binding , underpins the true power of FRET sensors as tools for cAMP measurement in living cells . Currently FRET sensors remain the tools o"
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    ABSTRACT: Intracellular 3 ′-5 ′-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming—all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP lifespan under tight control—something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental setups involving living cells, providing the guidance for rational selection of the best tools for particular needs.
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    • "On the other hand, the structural protein A kinase anchoring protein 149 (AKAP149) binds PDE4A and protein kinase A (PKA) to the outer mitochondrial membrane (Asirvatham et al., 2004; Carlucci et al., 2008). These three components make a complex that is regulated by cAMP levels, since this second messenger activates PKA, and the whole complex moves around the cell depending on cAMP gradients (Baillie et al., 2005; Sample et al., 2012). Since YTX modulates PDEs, the complex was studied after toxin treatment in the tumor K-562 cell line. "
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    ABSTRACT: Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed.
    Frontiers in Pharmacology 06/2015; 6:124. DOI:10.3389/fphar.2015.00124 · 3.80 Impact Factor
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    • "range by changing the FRET acceptor Citrine to a circularly permuted Venus at lysine 194 (cpV-L194; DiPilato and Zhang, 2009; Figure 1C). The large dynamic range of ICUE3 (∼100% emission ratio change) makes it suitable for subcellular targeting for detecting local cAMP changes (e.g., plasma membrane and nucleus (Sample et al., 2012), sarcoplasmic reticulum (Liu et al., 2012), primary cilia (Marley et al., 2013)) as addition of subcellular localization tags sometimes leads to decreased response amplitudes. The Jalink lab developed a similar biosensor CFP-Epac(δDEP- CD)-YFP using Epac1 149−881 flanked by an amino-terminal CFP and carboxy-terminal YFP (Ponsioen et al., 2004). "
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    ABSTRACT: The second messengers cAMP and cGMP transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks.
    Frontiers in Cellular Neuroscience 11/2014; 8(395). DOI:10.3389/fncel.2014.00395 · 4.29 Impact Factor
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