| Info. | Vol.11 - No.1 (2017.03.20) |
|---|---|
| Title | In situ PKA Activity Assay by Selective Detection of its Catalytic Subunit Using Antibody Arrays |
| Authors | Danishmalik Rafiq Sayyed1, Se-Hui Jung1, Min-Soo Kim1,2, Eun-Taek Han3, Won Sun Park4, Seok-Ho Hong5, Young-Myeong Kim1 & Kwon-Soo Ha1,* |
| Institutions | 1Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Republic of Korea 2Department of Anesthesiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Republic of Korea 3Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine,Chuncheon, Kangwon-Do 24341, Republic of Korea 4Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Republic of Korea 5Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Republic of Korea *Correspondence and requests for materials should be addressed to K.-S. Ha (ksha@kangwon.ac.kr) |
| Abstract | 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 welltype amine arrays. The limit of detection was 0.02 關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 in 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. |
| Keyword | In situ PKA activity, cPKA autophosphorylation, Antibody array, Forskolin, AGS cells, HUVECs |
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