Small-molecule FRET probes for protein kinase activity monitoring in living cells

In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system

Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.     

Compartmentalisation of cAMP and Ca(2+) signals

The available knowledge concerning second messengers such as Ca(2+) and cAMP has grown immensely in the past few years. The concept of tight spatial compartmentalisation of these signals within cells has led to more refined models of intracellular signalling. The development of recombinant probes based on the green fluorescent protein have allowed the monitoring of these second messenger levels in single cells, with high spatial and temporal resolution.     

Transgenic fruit-flies expressing a FRET-based sensor for in vivo imaging of cAMP dynamics

3'-5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous intracellular second messenger that mediates the action of various hormones and neurotransmitters and influences a plethora of cellular functions. In particular, multiple neuronal processes such as synaptic plasticity underlying learning and memory are dependent on cAMP signalling cascades. It is now well recognized that the specificity and fidelity of cAMP downstream effects are achieved through a tight temporal as well as spatial control of the cAMP signals. Approaches relying on real-time imaging and Fluorescence Resonance Energy Transfer (FRET)-based biosensors for direct visualization of cAMP changes as they happen in intact living cells have recently started to uncover the fine details of cAMP spatio-temporal signalling patterns. Here we report the generation of transgenic fruit-flies expressing a FRET-based, GFP-PKA sensor and their use in real-time optical recordings of cAMP signalling both ex vivo and in vivo in adult and developing organisms. These transgenic animals represent a novel tool for understanding the physiology of the cAMP signalling pathway in the context of a functioning body.     

Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.     

Phosphodiesterases and compartmentalized cAMP signalling in the heart

A key feature of the cAMP/cAMP-dependent protein kinase (PKA) transduction system is the compartmentalisation of its signalling enzymes and effectors. Given the large diversity of PKA targets within cardiac cells a precisely regulated and confined activity of such signalling pathway is essential for specificity of response. This appears to be achieved through the generation of local pools of high cAMP and activation of PKA at discrete subcellular locations. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP](i) in a stimulus-specific manner. A better understanding of the distinctive role that individual PDEs play in shaping the cAMP signal in heart cells may lead to the development of new strategies for selective pharmacologic manipulation of cAMP signalling in defined functional domains.     

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Previous work has shown that the protein kinase A (PKA)-regulated phosphodiesterase (PDE) 4D3 binds to A kinase-anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA-PDE4D3-AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals.