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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    • "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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    • "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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    • "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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