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.

Summary AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell vitality, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPKα1, not AMPKα2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.

The current complement of fluorescent proteins (FPs) contains color variants whose emission spectra span most of the visible spectrum, providing researchers with a versatile toolset of fluorescent probes for live cell imaging applications. FP family members generate their chromophores autocatalytically through a series of posttranslational modifications. The fluorescence characteristics of GFP-family members are influenced in important ways by the local microenvironment surrounding the chromophore. In this tutorial review, we first examine the molecular factors that influence the photophysical properties of FP family members and then briefly discuss some of the ways in which these fascinating proteins have been applied to the field of live cell imaging.

Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3− and/or H+ mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3−-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3− in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

In differentiated rodent hippocampal neurons, a FRET-based activity reporter reveals distinct spatiotemporal activity patterns of BRSK and AMPK, two homologous kinases that play important roles in neuronal polarity. AMPK exhibits maximal stimulated activity, whereas BRSK displays polarized basal activity in the distal region of the axon and axon tips.

In this Commentary, we discuss two sets of genetically encoded molecular tools that have significantly enhanced our ability to observe and manipulate complex biochemical processes in their native context and that have been essential in deepening our molecular understanding of how intracellular signaling networks function. In particular, genetically encoded biosensors are widely used to directly visualize signaling events in living cells, and we highlight several examples of basic biosensor designs that have enabled researchers to capture the spatial and temporal dynamics of numerous signaling molecules, including second messengers and signaling enzymes, with remarkable detail. Similarly, we discuss a number of genetically encoded biochemical perturbation techniques that are being used to manipulate the activity of various signaling molecules with far greater spatial and temporal selectivity than can be achieved using standard pharmacological or genetic techniques, focusing specifically on examples of chemically driven and light-inducible perturbation strategies. We then describe recent efforts to combine these diverse and powerful molecular tools into a unified platform that can be used to elucidate the molecular details of biological processes that may potentially extend well beyond the realm of signal transduction.

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3ζ recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3ζ. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with14-3-3ζ that lowers the substrate K m . This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3ζ heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3ζ-AANAT interaction using 14-3-3ζ fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3ζ interaction.Melatonin is a key circadian rhythm hormone that is produced in a diurnal pattern, driven by changes in the level of the biosynthetic enzyme serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] 1 (1,2). The neuroendocrine network responsible for modulating AANAT levels has been intensively investigated, and some aspects of this regulation are now understood. Light detected by the retina results in neurotransmission through the suprachiasmatic nucleus that in turn induces a drop in norepinephrine stimulation † Supported by the NIH. *To whom correspondence should be addressed. E-mail: E-mail: pcole@jhmi.edu. Telephone: (410) 614-8849. Fax: (410) 614-7717. 1 Abbreviations: PKA, protein kinase A; AANAT, arylalkylamine N-acetyltransferase or serotonin N-acetyltransferase; GST, glutathione S-transferase; FRET, fluorescence resonance energy transfer; HEPES, hydroxyethylpiperazineethanesulfonate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; NMM, N-methylmorpholine; MPAA, mercaptophenylacetic acid; MESNA, mercaptoethane sulfonate sodium; ss-WT, semisynthetic wild-type AANAT (see Figure 1); ss-pS205, semisynthetic AANAT containing a pSer at position 205 (see Figure 1); ss-pT31, semisynthetic AANAT containing a pSer at position 31 (see Figure 1); ss-pT31-pS205, semisynthetic AANAT containing a pThr at position 31 and a pSer at position 205 (see Figure 1); Δ-CD, C-terminally deleted recombinant AANAT amino acids 1-198; ss-GC-Insert, semisynthetic AANAT using C-terminal ligation and GC inserted between amino acids 33 a...

No abstract