@inproceedings{diskar2005investigating, author = {Diskar, M. and Herberg, F. W. and Prinz, A.}, interhash = {09f9a8a93396596add8cce2554129117}, intrahash = {4aa07ee13bfbe9fcad329cc1a584953b}, issn = {{1742-464X}}, journal = {FEBS JOURNAL}, month = jul, note = {{IUBMB 50th Anniversary Symposium, Budapest, HUNGARY, JUL 02-07, 2005}}, number = 1, pages = 303, times-cited = {{0}}, title = {Investigating human protein kinase X holoenzyme formation using bioluminescence resonance energy transfer (BRET)}, unique-id = {{ISI:000234826101555}}, volume = 272, year = 2005 } @article{soberg2012identification, abstract = {The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser(109) significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly(186) completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly(186) is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg(2+), which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser(109) or Gly(186) may be faced with abnormal development and possibly severe disease.}, author = {Soberg, K. and Larsen, A. C. and Diskar, M. and Backe, P. H. and Bjoras, M. and Jahnsen, T. and Laerdahl, J. K. and Rognes, T. and Herberg, F. W. and Skalhegg, B. S.}, doi = {10.1371/journal.pone.0034838}, interhash = {e0bf40aeaa52c110525104af6f4d9815}, intrahash = {52e132d9f8096e76f53932f18e753318}, journal = {PLoS One}, number = 4, pmid = {22514673}, title = {Identification and Characterization of Novel Mutations in the Human Gene Encoding the Catalytic Subunit Calpha of Protein Kinase A (PKA)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/22514673}, volume = 7, year = 2012 } @mastersthesis{diskar2005charakterisierung, author = {Diskar, M.}, interhash = {0d4d6d23b192bcd315759c6ad7e29a3b}, intrahash = {ed46522e9c7febedac3f59c2f4433200}, pages = 89, school = {Universität Kassel}, title = {In vivo Charakterisierung von PKA Isoformen in eukaryotischen Zellen}, year = 2005 } @phdthesis{diskar2009funktionelle, author = {Diskar, M.}, interhash = {e3835a6b214578124d39ba817edb3521}, intrahash = {a69baec20ef2de813432ea13ec777b6f}, pages = 198, school = {Universität Kassel}, title = {Funktionelle Charakterisierung von Proteinkinase A Isoformen in Eukaryoten}, year = 2009 } @article{Prinz:2008:Proteomics:18283669, abstract = {This review aims to provide an overview of current optical procedures used in functional proteomics, investigating protein localization, protein-protein interaction, intracellular signaling events, and second messenger generation in living cells. Reporter assays using proteins tagged with fluorescent or bioluminescent moieties are discussed. Recently, intracellular biosensor assays, flow cytometry-based techniques (fluorescent cell barcoding), as well as transfected cell microarray assays involving RNA interference coupled with automated imaging were introduced and have been adopted as screening platforms for annotating small molecules, investigating signaling events, or in phenotype analysis. These novel methodological advances include improved image acquisition and processing techniques and help linking in vitro observations to in vivo processes. In addition, the acquired data are increasingly quantitative in nature and will therefore pave the way for modeling of signaling cascades and other complex cellular events, an important step toward systems biology.}, author = {Prinz, A. and Reither, G. and Diskar, M. and Schultz, C.}, doi = {10.1002/pmic.200700802}, interhash = {084d252365dea438e1fa5a4862605cf6}, intrahash = {2361ad6c08383c4ebdcc8abe1c1df5fc}, journal = {Proteomics}, month = mar, number = 6, pages = {1179-1196}, pmid = {18283669}, title = {Fluorescence and bioluminescence procedures for functional proteomics}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18283669}, volume = 8, year = 2008 } @article{Farquhar:2008:J-Virol:18579596, abstract = {Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.}, author = {Farquhar, M. J. and Harris, H. J. and Diskar, M. and Jones, S. and Mee, C. J. and Nielsen, S. U. and Brimacombe, C. L. and Molina, S. and Toms, G. L. and Maurel, P. and Howl, J. and Herberg, F. W. and van Ijzendoorn, S. C. and Balfe, P. and McKeating, J. A.}, doi = {10.1128/JVI.00592-08}, interhash = {d3b291842a7e657cb2b3b398459c9aef}, intrahash = {bea64e3bbb0b69f522ab93b02bc25ad1}, journal = {J Virol}, month = sep, number = 17, pages = {8797-8811}, pmid = {18579596}, title = {Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18579596}, volume = 82, year = 2008 } @techreport{diskar2006quantifizierung, author = {Diskar, M. and Herberg, F. W. and Prinz, A.}, institution = {Biospektrum}, interhash = {a69aee83d077b05cf7f5b5dfaf759258}, intrahash = {0856ccb7202aec968bfcf14bb38d24f1}, journal = {Biospektrum}, number = 6, pages = {629-631}, title = {Quantifizierung intrazellulärer Protein-Protein-Wechselwirkungen}, year = 2006 } @article{a2006novel, abstract = {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.}, annote = {Prinz, Anke Diskar, Mandy Erlbruch, Andrea Herberg, Friedrich W Research Support, Non-U.S. Gov't England Cellular signalling Cell Signal. 2006 Oct;18(10):1616-25. Epub 2006 Mar 9.}, author = {Prinz, A. and Diskar, M. and Erlbruch, A. and Herberg, F. W.}, edition = {2006/03/10}, interhash = {ae3db140d8110c02dd0851515d550264}, intrahash = {b4e68ae0d20f704908625a90d5729cee}, isbn = {0898-6568 (Print) 0898-6568 (Linking)}, journal = {Cell Signal}, pages = {1616-25}, series = 10, title = {Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16524697}, volume = 18, year = 2006 } @article{m2007molecular, abstract = {Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P(0)-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P(0)-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P(0), whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P(0) from serine to alanine. In contrast, PKA-Calpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P(0): Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.}, annote = {Diskar, Mandy Zenn, Hans-Michael Kaupisch, Alexandra Prinz, Anke Herberg, Friedrich W Research Support, Non-U.S. Gov't England Cellular signalling Cell Signal. 2007 Oct;19(10):2024-34. Epub 2007 Jun 9.}, author = {Diskar, M. and Zenn, H. M. and Kaupisch, A. and Prinz, A. and Herberg, F. W.}, edition = {2007/07/07}, interhash = {905ec0de28587a5b387f4bf71bb7e0ec}, intrahash = {9da61a91663e60c3d79680608b9e9151}, isbn = {0898-6568 (Print) 0898-6568 (Linking)}, journal = {Cell Signal}, pages = {2024-34}, series = 10, title = {Molecular basis for isoform-specific autoregulation of protein kinase A}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17614255}, volume = 19, year = 2007 } @article{m2010regulation, abstract = {cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human Protein Kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIalpha or RIbeta but not by the substrate inhibitors RIIalpha or RIIbeta. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied Bioluminescence Resonance Energy Transfer (BRET) and Surface Plasmon Resonance (SPR) in combination with site-directed mutagenesis. We observed that the conserved alphaH-alphaI loop residue Arg283 in PrKX is a crucial for its RI over RII preference, as a Arg283Leu mutant was able to form a holoenzyme complex with wildtype RII subunits. Changing the corresponding alphaH-alphaI loop residue in PKA Calpha (Leu277Arg), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of two to four, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.}, annote = {Journal articleThe Journal of biological chemistryJ Biol Chem. 2010 Sep 6.}, author = {Diskar, M. and Zenn, H. M. and Kaupisch, A. and Kaufholz, M. and Brockmeyer, S. and Sohmen, D. and Berrera, M. and Zaccolo, M. and Boshart, M. and Herberg, F. W. and Prinz, A.}, edition = {2010/09/08}, interhash = {0752e43af49cef48fb102f2b639b28e6}, intrahash = {edf4d9acbb7435188f8d1807f6998eac}, isbn = {1083-351X (Electronic) 0021-9258 (Linking)}, journal = {J Biol Chem}, number = 46, pages = {35910-8}, title = {Regulation of cAMP-dependent protein kinases: the human Protein Kinase X (PrKX) reveals the role of the catalytic subunit {alpha}H-{alpha}I loop}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20819953}, volume = 285, year = 2010 } @article{a2006application, annote = {Prinz, Anke Diskar, Mandy Herberg, Friedrich W Research Support, Non-U.S. Gov't Review Germany Chembiochem : a European journal of chemical biology Chembiochem. 2006 Jul;7(7):1007-12.}, author = {Prinz, A. and Diskar, M. and Herberg, F. W.}, edition = {2006/06/07}, interhash = {855247c8b0cb3cf5aa244fa6f1081b05}, intrahash = {689f2babf1a7e311f2154952d502fdc0}, isbn = {1439-4227 (Print) 1439-4227 (Linking)}, journal = {Chembiochem}, pages = {1007-12}, series = 7, title = {Application of bioluminescence resonance energy transfer (BRET) for biomolecular interaction studies}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16755626}, volume = 7, year = 2006 } @article{a2009regulatory, abstract = {Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.}, annote = {Wojtal, Kacper A Diskar, Mandy Herberg, Friedrich W Hoekstra, Dick van Ijzendoorn, Sven C D Research Support, Non-U.S. Gov't United States The Journal of biological chemistry J Biol Chem. 2009 Jul 31;284(31):20773-80. Epub 2009 May 22.}, author = {Wojtal, K. A. and Diskar, M. and Herberg, F. W. and Hoekstra, D. and van Ijzendoorn, S. C.}, edition = {2009/05/26}, interhash = {734427862c7c2242b354f6d2d8af53b3}, intrahash = {0a9f721e8775d77b2317403ea0af637f}, isbn = {1083-351X (Electronic) 0021-9258 (Linking)}, journal = {J Biol Chem}, pages = {20773-80}, series = 31, title = {Regulatory subunit I-controlled protein kinase A activity is required for apical bile canalicular lumen development in hepatocytes}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19465483}, volume = 284, year = 2009 }