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In situ PKA activity assay by selective detection of its catalytic subunit using antibody arrays

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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 μg/ 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.

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References

  1. Kong, D.H. et al. A peptide array-based serological protein kinase A activity assay and its application in cancer diagnosis. Analyst 140, 6588–6594 (2015).

    Article  CAS  Google Scholar 

  2. Skalhegg, B.S. & Tasken, K. Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci. 5, D678–693 (2000).

    CAS  Google Scholar 

  3. Cheung, J. et al. Structural insights into mis-regulation of protein kinase A in human tumors. Proc. Natl. Acad. Sci. USA 112, 1374–1379 (2015).

    Article  CAS  Google Scholar 

  4. Sands, W.A. & Palmer, T.M. Regulating gene transcription in response to cyclic AMP elevation. Cell. Signal. 20, 460–466 (2008).

    Article  CAS  Google Scholar 

  5. Lefkimmiatis, K. & Zaccolo, M. cAMP signaling in subcellular compartments. Pharmacol. Ther. 143, 295–304 (2014).

    Article  CAS  Google Scholar 

  6. Gold, M.G., Gonen, T. & Scott, J.D. Local cAMP signaling in disease at a glance. J. Cell Sci. 126, 4537–4543 (2013).

    Article  CAS  Google Scholar 

  7. Czyzyk, T.A., Sikorski, M.A., Yang, L. & McKnight, G.S. Disruption of the RIIbeta subunit of PKA reverses the obesity syndrome of Agouti lethal yellow mice. Proc. Natl. Acad. Sci. USA 105, 276–281 (2008).

    Article  CAS  Google Scholar 

  8. Gerthoffer, W.T., Solway, J. & Camoretti-Mercado, B. Emerging targets for novel therapy of asthma. Curr. Opin. Pharmacol. 13, 324–330 (2013).

    Article  CAS  Google Scholar 

  9. Bernstein, H.G. et al. Increased density of AKAP5-expressing neurons in the anterior cingulate cortex of subjects with bipolar disorder. J. Psychiatr. Res. 47, 699–705 (2013).

    Article  Google Scholar 

  10. Ritschel, T. et al. Crystal structure analysis and in silico pKa calculations suggest strong pKa shifts of ligands as driving force for high-affinity binding to TGT. Chembiochem. 10, 716–727 (2009).

    Article  CAS  Google Scholar 

  11. Shimizu, N. et al. The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein. Biochemistry 45, 3542–3547 (2006).

    Article  CAS  Google Scholar 

  12. Kim, C., Xuong, N.H. & Taylor, S.S. Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA. Science 307, 690–696 (2005).

    Article  CAS  Google Scholar 

  13. Solari, C.A. et al. Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae. Biochem. J. 462, 567–579 (2014).

    Article  CAS  Google Scholar 

  14. Zhang, P. et al. Single Turnover Autophosphorylation Cycle of the PKA RIIbeta Holoenzyme. PLoS Biol. 13, e1002192 (2015).

    Article  Google Scholar 

  15. Iyer, G.H., Moore, M.J. & Taylor, S.S. Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase. J. Biol. Chem. 280, 8800–8807 (2005).

    Article  CAS  Google Scholar 

  16. Byrne, A.M., Elliott, C., Hoffmann, R. & Baillie, G.S. The activity of cAMP-phosphodiesterase 4D7 (PDE 4D7) is regulated by protein kinase A-dependent phosphorylation within its unique N-terminus. FEBS Lett. 589, 750–755 (2015).

    Article  CAS  Google Scholar 

  17. Paulucci-Holthauzen, A.A. & O’Connor, K.L. Use of pseudosubstrate affinity to measure active protein kinase A. Anal. Biochem. 355, 175–182 (2006).

    Article  CAS  Google Scholar 

  18. Morgan, A.G. et al. Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination. Assay Drug. Dev. Technol. 2, 171–181 (2004).

    Article  CAS  Google Scholar 

  19. Li, T., Liu, X., Liu, D. & Wang, Z. Sensitive detection of protein kinase A activity in cell lysates by peptide microarray-based assay. Anal. Chem. 85, 7033–7037 (2013).

    Article  CAS  Google Scholar 

  20. Tang, S. et al. Cyclic-AMP-dependent protein kinase (PKA) activity assay based on FRET between cationic conjugated polymer and chromophore-labeled peptide. Analyst 139, 4710–4716 (2014).

    Article  CAS  Google Scholar 

  21. Li, Y., Xie, W. & Fang, G. Fluorescence detection techniques for protein kinase assay. Anal. Bioanal. Chem. 390, 2049–2057 (2008).

    Article  CAS  Google Scholar 

  22. Kasari, M. et al. Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers. Anal. Biochem. 422, 79–88 (2012).

    Article  CAS  Google Scholar 

  23. Xu, X. et al. Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots. Anal. Chem. 83, 52–59 (2011).

    Article  CAS  Google Scholar 

  24. Allen, M.D. & Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys Res. Commun. 348, 716–721 (2006).

    Article  CAS  Google Scholar 

  25. Chen, Y., Saulnier, J.L., Yellen, G. & Sabatini, B.L. A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. Front. Pharmacol. 5, 56 (2014).

    Article  Google Scholar 

  26. Liu, S., Zhang, J. & Xiang, Y.K. FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes. Biochem. Biophys Res. Commun. 404, 581–586 (2011).

    Article  CAS  Google Scholar 

  27. Lim, C.J. et al. Integrin-mediated protein kinase A activation at the leading edge of migrating cells. Mol. Biol. Cell 19, 4930–4941 (2008).

    Article  CAS  Google Scholar 

  28. McKenzie, A.J., Campbell, S.L. & Howe, A.K. Protein kinase A activity and anchoring are required for ovarian cancer cell migration and invasion. PloS One 6, e26552 (2011).

    Article  CAS  Google Scholar 

  29. Moskaug, J.O., Carlsen, H. & Blomhoff, R. Noninvasive in vivo imaging of protein kinase A activity. Mol. Imaging 7, 35–41 (2008).

    CAS  Google Scholar 

  30. Shults, M.D., Janes, K.A., Lauffenburger, D.A. & Imperiali, B. A multiplexed homogeneous fluorescencebased assay for protein kinase activity in cell lysates. Nat. Methods 2, 277–283 (2005).

    Article  CAS  Google Scholar 

  31. Namkoong, S. et al. Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling. Cell. Signal. 21, 906–915 (2009).

    Article  CAS  Google Scholar 

  32. Cho, Y.S. et al. Extracellular protein kinase A as a cancer biomarker: its expression by tumor cells and reversal by a myristate-lacking Calpha and RIIbeta subunit overexpression. Proc. Natl. Acad. Sci. USA 97, 835–840 (2000).

    Article  CAS  Google Scholar 

  33. Pearce, L.R., Komander, D. & Alessi, D.R. The nuts and bolts of AGC protein kinases. Nat. Rev. Mol. Cell Biol. 11, 9–22 (2009).

    Article  Google Scholar 

  34. Ubersax, J.A. & Ferrell, J.E., Jr. Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol. 8, 530–541 (2007).

    Article  CAS  Google Scholar 

  35. Jeon, H.Y. et al. C-reactive protein as a parameter for defining normal blood samples in identification and evaluation of serological biomarkers. Biochip J. 9, 35–43 (2015).

    Article  CAS  Google Scholar 

  36. Kong, D.H. et al. Normalization using a tagged-internal standard assay for analysis of antibody arrays and the evaluation of serological biomarkers for liver disease. Anal. Chim. Acta 718, 92–98 (2012).

    Article  CAS  Google Scholar 

  37. Jung, J.W. et al. Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays. Biosens. Bioelectron. 24, 1469–1473 (2009).

    Article  CAS  Google Scholar 

  38. Sambrook, J. & Russell, D.W. The condensed protocols from Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 2006; p v, 800 p.

    Google Scholar 

  39. Manning, G. et al. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    Article  CAS  Google Scholar 

  40. Haubrich, B.A. & Swinney, D.C. Enzyme activity assays for protein kinases: Strategies to identify active substrates. Curr. Drug Discov. Technol. 13, 2–15 (2016).

    Article  CAS  Google Scholar 

  41. Lochner, A. & Moolman, J.A. The many faces of H89: a review. Cardiovasc. Drug Rev. 24, 261–274 (2006).

    Article  CAS  Google Scholar 

  42. Cvijic, M.E. et al. Extracellular catalytic subunit activity of the cAMP-dependent protein kinase in prostate cancer. Clin. Cancer Res. 6, 2309–2317 (2000).

    CAS  Google Scholar 

  43. Wang, H. et al. Extracellular activity of cyclic AMPdependent protein kinase as a biomarker for human cancer detection: Distribution characteristics in a normal population and cancer patients. Cancer Epidem. Biomar. 16, 789–795 (2007).

    Article  CAS  Google Scholar 

  44. Kita, T. et al. Extracellular cAMP-dependent protein kinase (ECPKA) in melanoma. Cancer Letters 208, 187–191 (2004).

    Article  CAS  Google Scholar 

  45. Yi, S.J. et al. [Ca2+]-dependent generation of intracellular reactive oxygen species mediates maitotoxininduced cellular responses in human umbilical vein endothelial cells. Mol. Cell 21, 121–128 (2006).

    CAS  Google Scholar 

  46. Jung, J.W. et al. Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays. Biosens. Bioelectron. 24, 1469–1473 (2009).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, 24341, Republic of Korea

    Danishmalik Rafiq Sayyed, Se-Hui Jung, Min-Soo Kim, Young-Myeong Kim & Kwon-Soo Ha

  2. Department of Anesthesiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, 24341, Republic of Korea

    Min-Soo Kim

  3. Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, 24341, Republic of Korea

    Eun-Taek Han

  4. Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, 24341, Republic of Korea

    Won Sun Park

  5. Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, 24341, Republic of Korea

    Seok-Ho Hong

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  1. Danishmalik Rafiq Sayyed
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  2. Se-Hui Jung
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Correspondence to Kwon-Soo Ha.

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Sayyed, D.R., Jung, SH., Kim, MS. et al. In situ PKA activity assay by selective detection of its catalytic subunit using antibody arrays. BioChip J 11, 57–66 (2017). https://doi.org/10.1007/s13206-016-1108-5

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  • DOI: https://doi.org/10.1007/s13206-016-1108-5

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