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Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone Cdc37

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Abstract

CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone Cdc37 on a conserved serine residue (Ser13) is essential for the function of Cdc37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829–2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117–38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065–4074, 2004]. We have recently produced an anti-[pSer13]-Cdc37 antibody which specifically recognizes Cdc37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690–5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-Cdc37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of Cdc37 on Ser13 by CK2 holoenzyme or CK2α, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous Cdc37 on Ser13, as revealed by anti-[pSer13]-Cdc37, and overexpression of either CK2α or CK2β subunit enhanced the Cdc37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of Cdc37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of Cdc37 on Ser13. These results show that the anti-[pSer13]-Cdc37 antibody can be a promising new tool to evaluate in vivo CK2 activity.

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Abbreviations

Hsp90:

Heat shock protein 90

Cdc37:

Cell division cycle 37

DMAT:

2-Dimethylamino-4, 5, 6, 7-tetrabromo-1H-benzimidazole

TBB:

4, 5, 6, 7-Tetrabromobenzotriazole

IGF-I:

Insulin-like growth factor-I

FCS:

Fetal calf serum

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Acknowledgments

We thank T. Sakabe for excellent technical assistance. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  1. Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Yoshihiko Miyata & Eisuke Nishida

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  1. Yoshihiko Miyata
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  2. Eisuke Nishida
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Correspondence to Yoshihiko Miyata.

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Miyata, Y., Nishida, E. Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone Cdc37. Mol Cell Biochem 316, 127–134 (2008). https://doi.org/10.1007/s11010-008-9818-1

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  • DOI: https://doi.org/10.1007/s11010-008-9818-1

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