Stokka Anne J, Gesellchen Frank, Carlson Cathrine R, Scott John D, Herberg Friedrich W, Taskén Kjetil
Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0317 Oslo, Norway.
Biochem J. 2006 Dec 15;400(3):493-9. doi: 10.1042/BJ20060962.
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.
蛋白激酶A（cAMP依赖性蛋白激酶或蛋白激酶A）的亚细胞定位由其R（调节）亚基与A激酶锚定蛋白（AKAPs）之间的蛋白质-蛋白质相互作用决定。在本文中，我们报告了一种用于表征基于AKAP的蛋白激酶A锚定肽竞争物的方法——放大发光邻近均相分析（AlphaScreen）的开发。在该分析中，典型的锚定破坏剂Ht31在与RII特异性和双特异性AKAP的RIIα亚型结合中具有高效竞争性（IC50值分别为1.4±0.2 nM和6±1 nM）。相比之下，RIα亚型与双特异性AKAP的结合竞争效率较低（IC50为156±10 nM）。对两种RI选择性锚定破坏剂RIAD（RI锚定破坏剂）和PV - 38的表征显示，RIAD（IC50为13±1 nM）的效力比PV - 38（IC50为304±17 nM）高20倍，并且不参与RIIα - AKAP相互作用的竞争。我们还观察到，当在体外混合各组成部分时，从预先形成的蛋白激酶A - AKAP复合物中置换RII的动力学以及Ht31对RII - AKAP复合物形成的竞争在数量级上有所不同。对于RIα - AKAP复合物，未观察到这种效力差异。因此，AlphaScreen分析可能被证明是详细表征各种蛋白激酶A - AKAP复合物的有价值工具。
