In situ PKA Activity Assay by Selective Detection of its Catalytic Subunit Using Antibody Arrays

초록

Protein kinase A (PKA) plays a pivotal role in various biological processes and the pathogenesis of several diseases. However, systematic investigation of PKA functions in cells and tissues is limited due to the lack of a suitable high-throughput in situ PKA activity assay. Here, we present an array-based in situ PKA activity assay that employs selective detection of the catalytic form of PKA (cPKA; active and autophosphorylated) using antibody arrays. Antibody arrays were fabricated by applying anti-cPKA antibody onto well type amine arrays. The limit of detection was 0.02 g/mL. We successfully applied this assay to determine changes in intracellular and extracellular PKA activities in human gastric adenocarcinoma (AGS) cells and human umbilical vein endothelial cells (HUVECs) treated with forskolin. Forskolin induced activation of intracellular PKA in a dose-dependent manner, and this PKA activation was inhibited by the potent PKA inhibitor H-89. Extracellular PKA activity was also elevated by forskolin in a dose-dependent manner in AGS cells, but this elevation was hardly detectable HUVECs. Thus, our antibody array-based in situ PKA activity assay is suitable for investigating the regulation and functions of intracellular and extracellular PKA in cells and tissues and has a potential for use in cancer diagnosis.

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