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 RIIα isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4±0.2 nM and 6±1 nM respectively). In contrast, RIα 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 RIIα–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 RIα–AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA–AKAP complexes.
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Research Article|
November 28 2006
Characterization of A-kinase-anchoring disruptors using a solution-based assay
Anne J. Stokka;
Anne J. Stokka
*Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0317 Oslo, Norway
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Frank Gesellchen;
Frank Gesellchen
†Abteilung für Biochemie, Fachbereich Naturwissenschaften, Universität Kassel, Heinrich-Plett-Strasse 40, 34109 Kassel, Germany
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Cathrine R. Carlson;
Cathrine R. Carlson
*Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0317 Oslo, Norway
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John D. Scott;
John D. Scott
‡Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Science University, Portland, OR 97239, U.S.A.
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Friedrich W. Herberg;
Friedrich W. Herberg
†Abteilung für Biochemie, Fachbereich Naturwissenschaften, Universität Kassel, Heinrich-Plett-Strasse 40, 34109 Kassel, Germany
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Kjetil Taskén
Kjetil Taskén
1
*Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0317 Oslo, Norway
1To whom correspondence should be addressed (email Kjetil.Tasken@biotek.uio.no).
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Publisher: Portland Press Ltd
Received:
June 26 2006
Revision Received:
August 30 2006
Accepted:
September 01 2006
Accepted Manuscript online:
September 01 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 400 (3): 493–499.
Article history
Received:
June 26 2006
Revision Received:
August 30 2006
Accepted:
September 01 2006
Accepted Manuscript online:
September 01 2006
Citation
Anne J. Stokka, Frank Gesellchen, Cathrine R. Carlson, John D. Scott, Friedrich W. Herberg, Kjetil Taskén; Characterization of A-kinase-anchoring disruptors using a solution-based assay. Biochem J 15 December 2006; 400 (3): 493–499. doi: https://doi.org/10.1042/BJ20060962
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