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Friedrich W Herberg

Publications and source records attributed to Friedrich W Herberg.

16 recordsLinked to original sources
PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗
PubMed2006

Characterization of A-kinase-anchoring disruptors using a solution-based assay.

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.

A Kinase Anchor Proteins↗
PubMed2006

PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases.

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Analysis of posttranslational modifications exemplified using protein kinase A.

With the completion of the major genome projects, one focus in biomedical research has shifted from the analysis of the rather static genome to the highly dynamic proteome. The sequencing of whole genomes did not lead to much anticipated insights into disease mechanisms; however, it paved the way for proteomics by providing the databases for protein identification by peptide mass fingerprints. The relative protein distribution within a cell or tissue is subject to change upon external and internal stimuli. Signal transduction events extend beyond a simple change in protein levels; rather they are governed by posttranslational modifications (PTMs), which provide a quick and efficient way to modulate cellular signals. Because most PTMs change the mass of a protein, they are amenable to analysis by mass spectrometry. Their investigation adds a level of functionality to proteomics, which can be expected to greatly aid in the understanding of the complex cellular machinery involved in signal transduction, metabolism, differentiation or in disease. This review provides an overview on posttranslational modifications exemplified on the model system cAMP-dependent protein kinase. Strategies for detection of selected PTMs are described and discussed in the context of protein kinase function.

Acylation↗
PubMed2006

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

A Kinase Anchor Proteins↗
PubMed2006

Quantification of cAMP antagonist action in vitro and in living cells.

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Animals↗
PubMed2006

Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET).

Homogeneous protein-protein interaction assays without the need of a separation step are an essential tool to unravel signal transduction events in live cells. We have established an isoform specific protein kinase A (PKA) subunit interaction assay based on bioluminescence resonance energy transfer (BRET). Tagging human Ralpha(I)-, Ralpha(II)-, as well as Calpha-subunits of PKA with Renilla luciferase (Rluc) as the bioluminescent donor or with green fluorescent protein (GFP2) as the energy acceptor, respectively, allows to directly probe PKA subunit interaction in living cells as well as in total cell extracts in order to study side by side PKA type I versus type II holoenzyme dynamics. Several novel, genetically encoded cAMP sensors and-for the first time PKA type I sensors-were generated. When C- and R-subunits are assembled to the respective holoenzyme complexes inside the cell, BRET occurs with a signal up to three times above the background. An increase of endogenous cAMP levels as well as treatment with the cAMP analog 8-Br-cAMP is reflected by a dose-dependent BRET signal reduction in cells expressing wild type proteins. In contrast to type II, the dissociation of the PKA type I holoenzyme complex was never complete in cells with maximally elevated cAMP levels. Both sensors dissociated completely upon treatment with 8-Br-cAMP after cell lysis, consistent with in vitro activation assays using holoenzymes assembled from purified PKA subunits. Interestingly, incubation of cells with the PKA antagonist Rp-8-Br-cAMPS leads to a significant BRET signal increase in cells expressing PKA type I or type II isoforms, indicating a stabilization of the holoenzyme complexes in vivo. Mutant RI subunits with reduced (hRIalpha-R210K) or abolished (hRIalpha-G200E/G324E) cAMP binding capability were studied to quantify maximal signal to noise ratios for the RI-BRET sensor. Utilizing BRET we demonstrate that PKA type II holoenzyme was rendered insensitive to beta-adrenergic receptor stimulation with isoproterenol when anchoring to the plasma membrane of COS-7 cells was disrupted by either using Ht31 peptide or by depletion of membrane cholesterol.

Animals↗
PubMed2006

Differential binding studies applying functional protein microarrays and surface plasmon resonance.

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

Biosensing Techniques↗
PubMed2005

Direct optical detection of protein-ligand interactions.

Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.

Binding, Competitive↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells.

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle.

A Kinase Anchor Proteins↗
PubMed2004

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.

Antibodies↗
PubMed2003

Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A.

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.

Amino Acid Sequence↗
PubMed2003

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.

Animals↗
PubMed2003

Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain.

In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser(364) by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.

CSK Tyrosine-Protein Kinase↗