%0 Journal Article %1 j2006characterization %A Stokka, A. J. %A Gesellchen, F. %A Carlson, C. R. %A Scott, J. D. %A Herberg, F. W. %A Tasken, K. %B 3 %D 2006 %J Biochem J %K Herberg myown %P 493-9 %T Characterization of A-kinase-anchoring disruptors using a solution-based assay %U http://www.ncbi.nlm.nih.gov/pubmed/16948636 %V 400 %X 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. %Z Stokka, Anne J Gesellchen, Frank Carlson, Cathrine R Scott, John D Herberg, Friedrich W Tasken, Kjetil Research Support, Non-U.S. Gov't England The Biochemical journal Biochem J. 2006 Dec 15;400(3):493-9. %7 2006/09/05 %@ 1470-8728 (Electronic) 0264-6021 (Linking)

@article{j2006characterization, abstract = {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.}, added-at = {2010-10-11T20:39:04.000+0200}, annote = {Stokka, Anne J Gesellchen, Frank Carlson, Cathrine R Scott, John D Herberg, Friedrich W Tasken, Kjetil Research Support, Non-U.S. Gov't England The Biochemical journal Biochem J. 2006 Dec 15;400(3):493-9.}, author = {Stokka, A. J. and Gesellchen, F. and Carlson, C. R. and Scott, J. D. and Herberg, F. W. and Tasken, K.}, biburl = {https://www.bibsonomy.org/bibtex/2e2eeec1e6dabec269d4cd42ef1ff1062/biochemie}, edition = {2006/09/05}, interhash = {f4d4ba4fde68d2c45cef3b0d62580281}, intrahash = {e2eeec1e6dabec269d4cd42ef1ff1062}, isbn = {1470-8728 (Electronic) 0264-6021 (Linking)}, journal = {Biochem J}, keywords = {Herberg myown}, pages = {493-9}, series = 3, timestamp = {2012-07-24T09:39:18.000+0200}, title = {Characterization of A-kinase-anchoring disruptors using a solution-based assay}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16948636}, volume = 400, year = 2006 }