Aufsatz in einer Fachzeitschrift
Characterization of A-kinase-anchoring disruptors using a solution based assay
Details zur Publikation
Autor(inn)en: | Stokka, A.; Gesellchen, F.; Carlson, C.; Scott, J.; Herberg, F.; Taskén, K. |
Publikationsjahr: | 2006 |
Zeitschrift: | Biochemical Journal |
Seitenbereich: | 493-499 |
Jahrgang/Band : | 400 |
ISSN: | 0264-6021 |
Zusammenfassung, 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 (TM)) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RII alpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4 +/- 0.2 nM and 6 +/- 1 nM respectively). In contrast, RI alpha 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 RII alpha-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 RI alpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.
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 (TM)) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RII alpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4 +/- 0.2 nM and 6 +/- 1 nM respectively). In contrast, RI alpha 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 RII alpha-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 RI alpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.
