Systematic interpretation of cyclic nucleotide binding studies using KinetXBase.

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA.

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.

Determination of kinetic data using surface plasmon resonance biosensors.

The use of biosensors employing surface plasmon resonance (SPR) provides excellent instrumentation for a label-free, real-time investigation of biomolecular interactions. A broad range of biological applications including antibody-antigen interactions can be analyzed. One major advantage of kinetic analysis using SPR-based biosensors is the option of determining separately distinct association and dissociation rate constants exceeding the classical steady-state analysis of biomolecules. Based on these data new possibilities for drug design, characterizing human pathogens, and the development of therapeutic antibodies can be achieved. The hardware of commercially available systems is described, practical step by step procedures are given, and possibilities and limitations of the technology are discussed.

Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity.

OBJECTIVE: The cardioprotective effects of trapidil on ischemic reperfused (I/R) rabbit hearts were studied. Recently, we had shown that trapidil might activate protein kinase A (PKA). In this study, we examined the exact mode of PKA stimulating activity of trapidil. Finally, we investigated the effect of trapidil on the phosphorylation state of phospholamban (PLB), a major PKA target in the heart and key regulator of Ca(2+) sequestration via the sarcoplasmic reticulum Ca(2+)-ATPase. METHODS: Langendorff-hearts of New Zealand White rabbits were perfused at constant volume and subjected to global low-flow ischemia for 2 h, followed by 1 h of reperfusion. Subsequently, hearts were used for Western blot analysis of PLB phosphorylation. Furthermore, three different regulatory subunits and one catalytic subunit of PKA were overexpressed in E. coli. These PKA subunits were purified and used in an in vitro assay system to test the impact of trapidil on PKA activities in the absence and presence of cAMP. RESULTS: I/R resulted in a significant increase in left ventricular end-diastolic pressure and creatine kinase efflux in the hearts. Trapidil (10 microM) prevented these alterations. Using recombinant cAMP-free PKA isoforms, it was found that trapidil specifically stimulated PKAII but only did so in the presence of small amounts of added cAMP. Furthermore, the PKA-dependent 16Ser phosphorylation of PLB was markedly reduced in I/R. Trapidil largely normalized the 16Ser phosphorylation of PLB. CONCLUSIONS: The data demonstrate cardioprotective actions of trapidil in I/R and show a PKAII-dependent cAMP sensitizing effect of the compound. They also indicate PKA-dependent PLB phosphorylation as a target, suggesting an improved Ca(2+) uptake by the sarcoplasmic reticulum. This action might be involved in the cardioprotective effects of trapidil.