Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Abstract

Understanding how specific 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 (sAC) 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 uncover the activation of a resident pool of 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 (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

Pharmacological Distinction between Soluble and Transmembrane Adenylyl Cyclases

“…This appreciation demanded revisions of the traditional model, in which cAMP is produced at the plasma membrane and diffused to its effector proteins distributed throughout the cell, into modern models, which posit that cAMP signaling occurs in independently regulated, intracellular compartments or microdomains (Dessauer, 2009;Houslay, 2010;Zaccolo, 2011). Within a compartment, cAMP can be produced locally, by a dedicated adenylyl cyclase (Bundey and Insel, 2004;Acin-Perez et al, 2009;Wachten et al, 2010;Willoughby et al, 2010;Zippin et al, 2010;Sample et al, 2012;Di Benedetto et al, 2013;Lefkimmiatis et al, 2013), and it can modulate the activity of a local effector, such as PKA tethered by an A kinase-anchoring protein (Dessauer, 2009). The sanctity of individual microdomains would be maintained by physical or enzymatic barriers, such as membranes (Rich et al, 2000;Acin-Perez et al, 2009;Di Benedetto et al, 2013) or PDEs (Houslay, 2010), which limit diffusion of the second messenger, as well as by the anchoring properties of A kinaseanchoring proteins (Dessauer, 2009), which keep the cyclase and effector proteins within the desired microdomains.…”

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

“…Whereas CID systems are generalizable and can be adapted for the control of several different enzymes and signaling pathways, other chemically induced perturbation systems are more narrowly applicable and are tailored to specific signaling pathways. For instance, we recently described a novel method for the spatiotemporal manipulation of cAMP using soluble adenylyl cyclase (SMICUS) that can be used to control the duration, magnitude and location of a cAMP signal (Sample et al, 2012). Classic techniques for manipulating cAMP concentrations usually produce large-scale variations in cAMP concentrations throughout the cell; however, our system utilizes a truncated version of soluble adenylyl cyclase (sAC) that retains the ability to catalytically produce cAMP from ATP and can be localized to different parts of the cell through tagging with endogenous targeting motifs.…”

“…cAMP) (Baillie, 2009;Cooper, 2003;Houslay, 2010;Stangherlin and Zaccolo, 2012), as well as through structural factors, such as cellular compartments and scaffold proteins (Dodge-Kafka et al, 2006), which establish local microdomains that contain varying concentrations of these molecules. To probe how local cAMP concentrations affect the specificity of cAMP-PKA signaling, we developed the aforementioned method for generating site-specific cAMP signals using soluble adenylyl cyclase (Sample et al, 2012). Using this method in conjunction with real-time imaging and mechanistic modeling, we were able to infer the existence of a pool of PKA holoenzyme that resides in the nucleus in HEK-293 cells.…”

“…Specifically, when cAMP signals that are produced in or around the nucleus passed a critical threshold, we unequivocally demonstrated the rapid activation of a resident nuclear pool of PKA. However, cAMP that is produced at the plasma membrane activates a cytoplasmic pool of PKA, which then enters the nucleus by diffusion, as evidenced by the slow responses of the PKA activity reporter (Sample et al, 2012).…”

Cross-regulation of Phosphodiesterase 1 and Phosphodiesterase 2 Activities Controls Dopamine-mediated Striatal α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking

“…This process requires the phosphorylation of GluA1-containing AMPARs by cyclic nucleotide-dependent kinases, making receptor trafficking tightly coupled to local cyclic nucleotide levels (5-10). 3Ј-5Ј cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade cAMP and cGMP, are essential in shaping the spatial and temporal dynamics of signaling by maintaining the compartmentalization of cyclic nucleotides to ensure effective signal propagation to downstream effectors (11)(12)(13)(14)(15) and thus are poised to be significant regulators of AMPAR trafficking in response to DA stimulation.…”