Molecular and Cellular Biochemistry

, Volume 316, Issue 1–2, pp 37–47 | Cite as

Biochemical characterization of CK2α and α′ paralogues and their derived holoenzymes: evidence for the existence of a heterotrimeric CK2α′-holoenzyme forming trimeric complexes

  • Birgitte B. Olsen
  • Tine Rasmussen
  • Karsten Niefind
  • Olaf-Georg Issinger


Altogether 2 holoenzymes and 4 catalytic CK2 constructs were expressed and characterized i.e. CK2α21−335β2; CK2α′-derived holoenzyme; CK2α1−335; MBP-CK2α′; His-tagged CK2α and His-tagged CK2α′. The two His-tagged catalytic subunits were expressed in insect cells, all others in Escherichia coli. IC50 studies involving the established CK2 inhibitors DMAT, TBBt, TBBz, apigenin and emodin were carried out and the Ki values calculated. Although the differences in the Ki values found were modest, there was a general tendency showing that the CK2 holoenzymes were more sensitive towards the inhibitors than the free catalytic subunits. Thermal inactivation experiments involving the individual catalytic subunits showed an almost complete loss of activity after only 2 min at 45°C. In the case of the two holoenzymes, the CK2α′-derived holoenzyme lost ca. 90% of its activity after 14 min, whereas CK2α21−335β2 only showed a loss of ca. 40% by this time of incubation. Gel filtration analyses were performed at high (500 mM) and low (150 mM) monovalent salt concentrations in the absence or presence of ATP. At 500 mM NaCl the CK2α′-derived holoenzyme eluted at a position corresponding to a molecular mass of 105 kDa which is significantly below the elution of the CK2α21−335β2 holoenzyme (145 kDa). Calmodulin was not phosphorylated by either CK2α21−335β2 or the CK2α′-derived holoenzyme. However, in the presence of polylysine only the CK2α21−335β2 holoenzyme could use calmodulin as a substrate such as the catalytic subunits, in contrast to the CK2α′-derived holoenzyme which only phosphorylated calmodulin weakly. This attenuation may be owing to a different structural interaction between the catalytic CK2α′ subunit and non-catalytic CK2β subunit.


Protein kinase CK2 Isozymes Inhibitors Substrate 



Adenylyl imidodiphosphate




Maltose binding protein







We thank Drs. B. Boldyreff and B. Guerra for critically reading the manuscript and Hans H. Jensen for expert technical assistance. B.B.O is supported by grant no. DP06083 from the Danish Cancer Society, K.N from the Deutsche Forschungsgemeinschaft (DFG) grant no. NI 643/1-3 and O.G.I by grant no. 002521109210 from the Danish Cancer Society; grant no. 21-04-0517 from the Danish Research Agency, the NOVO Nordisk Foundation and from a donation dedicated to Cancer Research from Karen Marie Maaløe.


  1. 1.
    Issinger OG (1993) Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol Ther 59:1–30. doi:10.1016/0163-7258(93)90039-G PubMedCrossRefGoogle Scholar
  2. 2.
    Guerra B, Issinger OG (1999) Protein kinase CK2 and its role in cellular proliferation, development and pathology. Electrophoresis 20:391–408. doi:10.1002/(SICI)1522-2683(19990201)20:2<391::AID-ELPS391>3.0.CO;2-NPubMedCrossRefGoogle Scholar
  3. 3.
    Guerra B, Boldyreff B, Sarno S, Cesaro L, Issinger OG, Pinna LA (1999) CK2: a protein kinase in need of control. Pharmacol Ther 82:303–313. doi:10.1016/S0163-7258(98)00064-3 PubMedCrossRefGoogle Scholar
  4. 4.
    Allende CC, Allende JE (1999) Promiscuous subunit interaction: a possible mechanism for the regulation of protein kinase CK2. J Cell Biochem 30:129–136Google Scholar
  5. 5.
    Pinna LA (2002) Protein kinase CK2: a challenge to canons. J Cell Sci 115:32873–33878. doi:10.1242/jcs.00074 CrossRefGoogle Scholar
  6. 6.
    Litchfield DW (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 369:1–15. doi:10.1042/BJ20021469 PubMedCrossRefGoogle Scholar
  7. 7.
    Olsen BB, Niefind K, Issinger OG (2005) Inter-and supramolecular interactions of protein kinase CK2 and their relevance for genome integrity. Genome integrity: Facets and perspectives, vol 1. Springer-Verlag Berl Heidelberg, pp 315–342Google Scholar
  8. 8.
    Meggio F, Pinna LA (2003) One-thousand-and-one substrates for protein kinase CK2? FASEB J 173:349–368. doi:10.1096/fj.02-0473rev CrossRefGoogle Scholar
  9. 9.
    Niefind K, Pütter B, Guerra B, Issinger OG, Schomburg D (1999) GTP plus water mimic ATP in the active site of protein kinase CK2. Nat Struct Biol 6:1100–1103. doi:10.1038/70033 PubMedCrossRefGoogle Scholar
  10. 10.
    Lozeman FJ, Litchfield DW, Pienning C, Takio K, Walsh KA, Krebs ED (1990) Isolation and characterization of human clones encoding the α or α′ subunits of casein kinase II. Biochemistry 29:8436–8447. doi:10.1021/bi00488a034 PubMedCrossRefGoogle Scholar
  11. 11.
    Guerra B, Siemer S, Boldyreff B, Issinger OG (1999) Protein kinase CK2: evidence for a protein kinase CK2β subunit fraction, devoid of the catalytic CK2α subunit, in mouse brain and testicles. FEBS Lett 462:353–357. doi:10.1016/S0014-5793(99)01553-7 PubMedCrossRefGoogle Scholar
  12. 12.
    Xu X, Toselli PA, Russell LD, Seldin DC (1999) Globozoospermia in mice lacking the casein kinase II α′ subunit. Nat Genet 23:118–121. doi:10.1038/12729 PubMedCrossRefGoogle Scholar
  13. 13.
    Lou DY, Dominguez I, Toselli P, Landesman-Bollag E, O’Brien C, Seldin DC (2008) The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development. Mol Cell Biol 28:131–139. doi:10.1128/MCB.01119-07 PubMedCrossRefGoogle Scholar
  14. 14.
    Buchou T, Vernet M, Blond O, Jensen HH, Pointu H, Olsen BB et al (2003) Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol Cell Biol 23:908–915. doi:10.1128/MCB.23.3.908-915.2003 PubMedCrossRefGoogle Scholar
  15. 15.
    Münstermann U, Fritz G, Seitz G, Lu YP, Schneider HR, Issinger OG (1990) Casein kinase II is elevated in solid human tumors and rapidly proliferating non-neoplastic tissue. Eur J Biochem 189:251–251. doi:10.1111/j.1432-1033.1990.tb15484.x PubMedCrossRefGoogle Scholar
  16. 16.
    Ahmed K, Davis AT, Wang H, Faust RA, Yu S, Tawfic S (2000) Significance of protein kinase CK2 nuclear signaling in neoplasia. J Cell Biochem Suppl 35:130–135. doi:10.1002/1097-4644(2000)79:35+<130::AID-JCB1136>3.0.CO;2-NPubMedCrossRefGoogle Scholar
  17. 17.
    Tawfic S, Yu S, Wang H, Faust RA, Davis A, Ahmed K (2001) Protein kinase CK2 signal in neoplasia. Histol Histopathol 16:573–582PubMedGoogle Scholar
  18. 18.
    Pagano MA, Cesaro L, Meggio F, Pinna LA (2006) Protein kinase CK2: a newcomer in the ‘druggable kinome’. Biochem Soc Trans 34:1303–1306. doi:10.1042/BST0341303 PubMedCrossRefGoogle Scholar
  19. 19.
    Niefind K, Guerra B, Pinna LA, Issinger OG, Schomburg D (1998) Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 Å resolution. EMBO J 17:2451–2462. doi:10.1093/emboj/17.9.2451 PubMedCrossRefGoogle Scholar
  20. 20.
    Niefind K, Guerra B, Ermakowa I, Issinger OG (2001) Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme. EMBO J 20:5320–5331. doi:10.1093/emboj/20.19.5320 PubMedCrossRefGoogle Scholar
  21. 21.
    Olsen BB, Boldyreff B, Niefind K, Issinger OG (2006) Purification and characterization of the CK2α′-based holoenzyme, an isozyme of CK2α: a comparative analysis. Protein Expr Purif 47:651–661. doi:10.1016/j.pep. 2005.12.001 PubMedCrossRefGoogle Scholar
  22. 22.
    Ermakova I, Boldyreff B, Issinger OG, Niefind K (2003) Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit. J Mol Biol 330:925–934. doi:10.1016/S0022-2836(03)00638-7 PubMedCrossRefGoogle Scholar
  23. 23.
    Grankowski N, Boldyreff B, Issinger OG (1991) Isolation and characterization of recombinant human casein kinase II subunits α and β from bacteria. Eur J Biochem 198:25–30. doi:10.1111/j.1432-1033.1991.tb15982.x PubMedCrossRefGoogle Scholar
  24. 24.
    Boldyreff B, Meggio F, Pinna LA, Issinger OG (1993) Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits. Biochemistry 32:12672–12677. doi:10.1021/bi00210a016 PubMedCrossRefGoogle Scholar
  25. 25.
    Guerra B, Niefind K, Ermarkova I, Issinger OG (2001) Characterization of CK2 holoenzyme variants with regard to crystallization. Mol Cell Biochem 227:3–11. doi:10.1023/A:1013184000557 PubMedCrossRefGoogle Scholar
  26. 26.
    Pagano MA, Meggio F, Ruzzene M, Andrzejewska M, Kazimierczuk Z, Pinna LA (2004) 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2. Biochem Biophys Res Commun 321:1040–1044. doi:10.1016/j.bbrc.2004.07.067 PubMedCrossRefGoogle Scholar
  27. 27.
    Pagano MA, Andrzejewska M, Ruzzene M, Sarno S, Cesar L, Bain J et al (2004) Optimization of protein kinase CK2 inhibitors derived from 4,5,6,7-tetrabromobenzimidazole. J Med Chem 47:6239–6247. doi:10.1021/jm049854a PubMedCrossRefGoogle Scholar
  28. 28.
    Meggio F, Pagano MA, Moro S, Zagotto G, Ruzzene M, Sarno S et al (2004) Inhibition of protein kinase CK2 by condensed polyphenolic derivatives. An in vitro and in vivo study. Biochemistry 43:12931–12936. doi:10.1021/bi048999g PubMedCrossRefGoogle Scholar
  29. 29.
    Knight ZA, Shokat KM (2005) Features of selective kinase inhibitors. Chem Biol 12:621–637. doi:10.1016/j.chembiol.2005.04.011 PubMedCrossRefGoogle Scholar
  30. 30.
    Zien P, Duncan JS, Skierski J, Bretner M, Litchfield DW, Shugar D (2005) Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: evaluation of their effects on cells and different molecular forms of CK2. Biochim Biophys Acta 1754:271–280PubMedGoogle Scholar
  31. 31.
    Sarno S, de Moliner E, Ruzzene M, Pagano MA, Battistutta R, Bain J et al (2003) Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo–5, 6-dihydroindolo-(1, 2-a)quinazolin-7-yl]acetic acid (IQA). Biochem J 374:639–646. doi:10.1042/BJ20030674 PubMedCrossRefGoogle Scholar
  32. 32.
    Sarno S, Salvi M, Battistutta R, Zanotti G, Pinna LA (2005) Features and potentials of ATP-site directed CK2 inhibitors. Biochim Biophys Acta 1754:263–270PubMedGoogle Scholar
  33. 33.
    Sarno S, Ruzzene M, Frascella P, Pagano MA, Meggio F, Zambon A et al (2005) Development and exploitation of CK2 inhibitors. Mol Cell Biochem 274:69–76. doi:10.1007/s11010-005-3079-z PubMedCrossRefGoogle Scholar
  34. 34.
    Nie Z, Perretta C, Erickson P, Margosiak S, Lu J, Averill A et al (2008) Structure-based design and synthesis of novel macrocyclic pyrazolo[1, 5a][1, 3, 5]triazine compounds as potent inhibitors of protein kinase CK2 and their anticancer activities. Bioorg Med Chem Lett 18:619–623. doi:10.1016/j.bmcl.2007.11.074 PubMedCrossRefGoogle Scholar
  35. 35.
    Persson T, Yde CW, Rasmussen JE, Rasmussen TL, Guerra B, Issinger OG et al (2007) Pyrazole carboxamides and carboxylic acids as protein kinase inhibitors in aberrant eukaryotic signal transduction: induction of growth arrest in MCF-7 cancer cells. Org Biomol Chem 5:3963–3970. doi:10.1039/b711279c PubMedCrossRefGoogle Scholar
  36. 36.
    Golub AG, Yakovenko OY, Prykhod’ko AO, Lukashov SS, Bdzhola VG, Yarmoluk SM (2007) Evaluation of 4,5,6,7-tetrahalogeno-1H-isoindole-1, 3(2H)-diones as inhibitors of human protein kinase CK2. Biochim Biophys Acta 1784:143–149PubMedGoogle Scholar
  37. 37.
    Filhol O, Cochet C (2007) Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction. Biochem J 408:363–373. doi:10.1042/BJ20070825 PubMedCrossRefGoogle Scholar
  38. 38.
    Nie Z, Perreta C, Erickson P, Margosia S, Almassy R, Lu J et al (2007) Structure-based design, synthesis, and study of pyrazolo[1, 5-a][1, 3, 5]triazine derivatives as potent inhibitors of protein kinase CK2. Bioorg Med Chem Lett 17:4191–4195. doi:10.1016/j.bmcl.2007.05.041 PubMedCrossRefGoogle Scholar
  39. 39.
    Raaf J, Klopffleisch K, Issinger OG, Niefind K (2008) The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin. J Mol Biol. doi:10.1016/j.jmb.2008.01.008
  40. 40.
    Meggio F, Boldyreff B, Marin O, Marchiori F, Perich JW, Issinger OG et al (1992) The effect of polylysine on casein kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme. Eur J Biochem 205:939–945. doi:10.1111/j.1432-1033.1992.tb16860.x PubMedCrossRefGoogle Scholar
  41. 41.
    Romero-Oliva F, Jacob G, Allende JE (2003) Dual effect of lysine-rich polypeptides on the activity of protein kinase CK2. J Cell Biochem 89:348–355. doi:10.1002/jcb.10493 PubMedCrossRefGoogle Scholar
  42. 42.
    Arrigoni G, Marin O, Pagano MA, Settimo L, Paolin B, Meggio F et al (2004) Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences. Biochemistry 43:12788–12798. doi:10.1021/bi049365c PubMedCrossRefGoogle Scholar
  43. 43.
    Dobrowolska G, Lozeman FJ, Donxia L, Krebs EG (1999) CK2, a protein kinase of the next millenium. Mol Cell Biochem 191:3–12. doi:10.1023/A:1006882910351 PubMedCrossRefGoogle Scholar
  44. 44.
    Issinger OG, Brockel C, Boldyreff B, Pelton JT (1992) Characterization of the α and β subunits of casein kinase 2 by Far-UV CD spectroscopy. Biochemistry 31:6098–6103. doi:10.1021/bi00141a020 PubMedCrossRefGoogle Scholar
  45. 45.
    Glover CV (1986) A filamentous form of drosophila casein kinase II. J Biol Chem 261:14349–14354PubMedGoogle Scholar
  46. 46.
    Valero E, DeBonis S, Filhol O, Wade H, Langowski J, Chambaz EM et al (1995) Quartenary structure of casein kinase 2: Characterization of multiple oligomeric states and relation with its catalytic activity. J Biol Chem 270:8345–8352. doi:10.1074/jbc.270.14.8345 PubMedCrossRefGoogle Scholar
  47. 47.
    Pagano MA, Sarno S, Poletto G, Cozza G, Pinna LA, Meggio F (2005) Autophosphorylation at the regulatory β-subunit reflects the supramolecular organization of protein kinase CK2. Mol Cell Biochem 274:23–29. doi:10.1007/s11010-005-3116-y PubMedCrossRefGoogle Scholar
  48. 48.
    Litchfield DW, Lozeman FJ, Cicirelle MF, Harrylock M, Ericsson LH, Piening CJ et al (1991) Phosphorylation of the β subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2. J Biol Chem 266:20380–20389PubMedGoogle Scholar
  49. 49.
    Boldyreff B, James P, Staudenmann W, Issinger OG (1993) Ser-2 is the autophosphorylation site in the β subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 β. Eur J Biochem 218:515–521. doi:10.1111/j.1432-1033.1993.tb18404.x PubMedCrossRefGoogle Scholar
  50. 50.
    Niefind K, Issinger OG (2005) Primary and secondary interactions between CK2α and CK2β lead to ring-like structures in the crystals of the CK2 holoenzyme. Mol Cell Biochem 274:3–14. doi:10.1007/s11010-005-3114-0 PubMedCrossRefGoogle Scholar
  51. 51.
    Poole A, Poore T, Bandhakavi S, McCann RO, Hanna DE, Glover CV (2005) A global view of CK2 function and regulation. Mol Cell Biochem 274:163–170. doi:10.1007/s11010-005-2945-z PubMedCrossRefGoogle Scholar
  52. 52.
    Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA, USAGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Birgitte B. Olsen
    • 1
  • Tine Rasmussen
    • 1
  • Karsten Niefind
    • 2
  • Olaf-Georg Issinger
    • 1
  1. 1.Institut for Biokemi og Molekylær BiologiSyddansk UniversitetOdenseDenmark
  2. 2.Department für Chemie, Institut für BiochemieUniversität zu KölnKölnGermany

Personalised recommendations