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Hanke S.E.,University of Kassel | Bertinetti D.,University of Kassel | Badel A.,University of Kassel | Schweinsberg S.,University of Kassel | And 2 more authors.
New Biotechnology | Year: 2011

cAMP (Adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network. © 2010 Elsevier B.V.


Wolter S.,Hannover Medical School | Dove S.,University of Regensburg | Golombek M.,Hannover Medical School | Schwede F.,BIOLOG Life Science Institute | Seifert R.,Hannover Medical School
Naunyn-Schmiedeberg's Archives of Pharmacology | Year: 2014

There is increasing evidence for a role of cytidine 3′,5′-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin- cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N4-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N4-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG. © 2014 Springer-Verlag Berlin Heidelberg.


Brunskole Hummel I.,Hannover Medical School | Brunskole Hummel I.,University of Regensburg | Reinartz M.T.,Hannover Medical School | Kalble S.,Hannover Medical School | And 4 more authors.
PLoS ONE | Year: 2013

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2 •-) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2 •- formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2 •- production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2 •- production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2 •- assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2 •- production after β2AR-stimulation although cAMP-increasing substances inhibited O2 •- production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2 •- production in a cAMP-independent manner. © 2013 Brunskole Hummel et al.


Stokman G.,Leiden University | Qin Y.,Leiden University | Genieser H.-G.,BIOLOG Life Science Institute | Schwede F.,BIOLOG Life Science Institute | And 5 more authors.
Journal of the American Society of Nephrology | Year: 2011

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2′-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2′-O-MecAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2′-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2′-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of β-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation. Copyright © 2011 by the American Society of Nephrology.


Borner S.,University of Wurzburg | Schwede F.,BIOLOG Life Science Institute | Schlipp A.,University of Wurzburg | Berisha F.,University of Wurzburg | And 4 more authors.
Nature Protocols | Year: 2011

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d. © 2011 Nature America, Inc. All rights reserved.

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