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Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET2

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Abstract

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET2 pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP2. This BRET2 sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET2 sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET2 signal by up to 50 %. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50 %. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET2 sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

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Acknowledgments

The work was supported by Grants from the Danish Cancer Society (OGI 252-1109-210), the Danish Research Council (OGI 21-04-0517), the Slovenian Research Agency program (M. Vrecl P4-0053), and the Deutsche Forschungsgemeinschaft (NI 643/4-1). We thank Hans H. Jensen for cloning of CK2α-MycHis.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

    Gerda M. Hübner, Barbara Guerra & Olaf-Georg Issinger

  2. Department of Incretin & Islet Biology, Novo Nordisk A/S, Måløv, Denmark

    Jane Nøhr Larsen

  3. Department of Chemistry, Institute for Biochemistry, University of Cologne, Cologne, Germany

    Karsten Niefind

  4. Veterinary Faculty, Institute of Anatomy, Histology & Embryology, University of Ljubljana, Ljubljana, Slovenia

    Milka Vrecl

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  1. Gerda M. Hübner
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  2. Jane Nøhr Larsen
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  3. Barbara Guerra
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  6. Olaf-Georg Issinger
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Correspondence to Olaf-Georg Issinger.

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Hübner, G.M., Larsen, J.N., Guerra, B. et al. Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET2 . Mol Cell Biochem 397, 285–293 (2014). https://doi.org/10.1007/s11010-014-2196-y

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