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.
Stokka
Anne J
AJ
Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0317 Oslo, Norway.
Gesellchen
Frank
F
Carlson
Cathrine R
CR
Scott
John D
JD
Herberg
Friedrich W
FW
Taskén
Kjetil
K
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011506:Proteins / Q000378:metabolism
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C400224:Ht 31 protein, synthetic; D021122:Protein Subunits; D011506:Proteins; D017868:Cyclic AMP-Dependent Protein Kinases