Skip to main content
SpringerLink
Log in

Biochemical characterization of the recombinant human Drosophila homologues Timekeeper and Andante involved in the Drosophila circadian oscillator

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

The Drosophila clock proteins timekeeper (CK2αTik) and andante (CK2βAnd) are mutated CK2α and CK2β subunits, respectively.

In order to revisit the hypothesis concerning a perturbation of the β/β and/or α/β subunit association, involving the andante mutant we have cloned, expressed and purified the recombinant andante mutant CK2βAnd and a CK2 holoenzyme composed of CK2βAnd and the wildtype CK2α subunit. Biochemical analyses using gel filtration analysis, inhibitor and heat treatment, as well as urea denaturation studies did not yield significant differences between the wildtype holoenzyme (α2β2) and a holoenzyme containing wildtype CK2α and andante CK2βAnd.

The timekeeper mutant, CK2αTik has been reported to show a significant reduction in enzyme activity. In order to closely investigate the reason for this reduction in activity, we have also cloned and expressed the human homologue of Drosophila timekeeper. Using a CK2 holoenzyme containing the human timekeeper mutant and the wildtype CK2β subunit we could confirm a strongly reduced activity towards CK2 substrates, but also a significant reduction in the autophosphorylation of the CK2β in the absence of any substrate. Based on a structure-based model we postulate that the mutation M161K in Drosophila (i.e. M163K in human) is responsible for the drastic loss of activity, where the lysine residue may cause improper binding of the tri-nucleotide.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Guerra B, Issinger OG: Protein kinase CK2 and its role in cellular proliferation, development and pathology. Electrophoresis 20: 391–408, 1999

    PubMed  Google Scholar 

  2. Pinna LA: Protein kinase CK2: a challenge to canons. J Cell Sci 115: 3873–3878, 2002

    PubMed  Google Scholar 

  3. Ahmed K, Gerber DA, Cochet C: Joining the cell survival squad: an emerging role for protein kinase CK2. Trends Cell Biol 12: 226–230, 2002

    PubMed  Google Scholar 

  4. Litchfield DW: Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 369: 1–15, 2003

    PubMed  Google Scholar 

  5. Niefind K, Guerra B, Pinna LA, Issinger OG, Schomburg D: Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 Å resolution. EMBO J 17: 2451–2462, 1998

    PubMed  Google Scholar 

  6. Niefind K, Putter M, Guerra B, Issinger OG, Schomburg D: GTP plus water mimic ATP in the active site of protein kinase CK2. Nat Struct Biol 12: 1100–1103, 1999

    Google Scholar 

  7. Chantalat L, Leroy D, Filhol O, Nueda A, Benitez MJ, Chambaz EM, Cochet C, Dideberg O: Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization. EMBO J 18: 2930–2940, 1999

    PubMed  Google Scholar 

  8. Niefind K, Guerra B, Ermakowa I, Issinger OG: Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme. EMBO J 20: 5320–5331, 2001

    PubMed  Google Scholar 

  9. Münstermann U, Fritz G, Seitz G, Lu YP, Schneider HR, Issinger OG: Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue. Eur J Biochem 189: 251–257, 1990

    PubMed  Google Scholar 

  10. Tawfic S, Yu S, Wang H, Faust R, Davis A, Ahmed K: Protein kinase CK2 signal in neoplasia. Histol Histophathol 16: 573–582, 2001

    Google Scholar 

  11. Seldin DC, Leder P: Casein kinase II alpha transgene-induced murine lymphoma: relation to theilerosis in cattle. Science 267: 894–897, 1995

    PubMed  Google Scholar 

  12. Xu X, Toselli PA, Russell LD, Seldin DC: Globozoospermia in mice lacking the casein kinase II alpha’ catalytic subunit. Nat Genet 1: 118–121, 1999

    Google Scholar 

  13. Padmanabha R, Chen-Wu JL, Hanna DE, Glover CV: Isolation, sequencing, and disruption of the yeast CKA2 gene: Casein kinase II is essential for viability in Saccharomyces cerevisiae. Mol Cell Biol 10: 4089–4099, 1990

    PubMed  Google Scholar 

  14. Buchou T, Vernet M, Blond O, Jensen HH, Pointu H, Olsen BB, Cochet C, Issinger OG, Boldyreff B: Disruption of the regulatory β subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol Cell Biol 23: 908–915, 2003

    PubMed  Google Scholar 

  15. Meggio F, Pinna LA: One-thousand-and-one substrates of protein kinase CK2. FASEB J 17: 349–368, 2003

    PubMed  Google Scholar 

  16. Riera M, Figueras M, Lopez C, Goday A, Pages M: Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize. Proc Natl Acad Sci USA 101: 9879–9884, 2004

    PubMed  Google Scholar 

  17. Yan Y, Chen P, He Q, Wang L, Liu Y: Phosphorylation by casein kinase II is necessary for the function of the Neurospora circadian clock. Mol Cell Biol 23: 6221–6228, 2003

    PubMed  Google Scholar 

  18. Lin JM, Kilman VL, Keegan K, Paddock B, Emery-Le M, Rosbach M, Allada R: A role for casein kinase 2a in the Drosophila circadian clock. Nature 420: 816–820, 2002

    PubMed  Google Scholar 

  19. Akten B, Jauch E, Genova GK, Kim EY, Edery I, Raabe T, Jackson FR: A role for CK2 in the Drosophila circadian oscillator. Nat Neurosci 6: 251–257, 2003

    PubMed  Google Scholar 

  20. Ermakova I, Boldyreff B, Issinger OG, Niefind K: Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit. J Mol Biol 330: 925–934, 2003

    PubMed  Google Scholar 

  21. Grankowski N, Boldyreff B, Issinger OG: Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria. Eur J Biochem 198: 25–30, 1991

    PubMed  Google Scholar 

  22. Schomburg D, Reichelt J: BRAGI: a comprehensive protein modelling program system. J Mol Graphics 6: 161–165, 1988

    Google Scholar 

  23. Blau J: A new role for an old kinase CK2 and the circadian clock. Nat Neurosci 6: 208–210, 2003

    PubMed  Google Scholar 

  24. Guerra B, Siemer S, Boldyreff B, Issinger OG: Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles. FEBS Lett 462: 353–357, 1999

    PubMed  Google Scholar 

  25. Toh KL, Jones RJ, Yan H, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ, Fu Y-H: An hPER2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291: 1040–1043, 2001

    PubMed  Google Scholar 

  26. Lenox RH, Gould TD, Manji HK: Endophentypes in bipolar disorder. Am J Med Genet 114: 391–406, 2002

    PubMed  Google Scholar 

  27. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE: The protein data bank. Nucl Acids Res 28: 235–242, 2000

    PubMed  Google Scholar 

  28. Esnouf RM: An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J Mol Graph Model 15: 132–134, 1997

    PubMed  Google Scholar 

  29. Merritt EA, Bacon DJ: Raster3D: photorealistic molecular graphics. Method Enzymol 277: 505–524, 1997

    Google Scholar 

  30. Boeckmann B, Bairoch A, Apweiler R, Blatter M-C, Estreicher A, Gasteiger E, Martin MJ, Michoud K, O’Donovan C, Phan I, Pilbout S, Schneider M: The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucl Acids Res 31: 365–370, 2003

    PubMed  Google Scholar 

  31. Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA, USA

Download references

Author information

Authors and Affiliations

  1. BMB, Syddansk Universitet, Odense, Denmark

    Tine Rasmussen, Iben H. E. Skjøth, Hans H. Jensen & Olaf-Georg Issinger

  2. Institut für Biochemie, Universität zu Köln, Germany

    Karsten Niefind

  3. KinaseDetect, ApS, Odense, Denmark

    Brigitte Boldyreff

  4. BMB, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

    Olaf-Georg Issinger

Authors
  1. Tine Rasmussen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Iben H. E. Skjøth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hans H. Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karsten Niefind
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brigitte Boldyreff
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olaf-Georg Issinger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Olaf-Georg Issinger.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rasmussen, T., Skjøth, I.H.E., Jensen, H.H. et al. Biochemical characterization of the recombinant human Drosophila homologues Timekeeper and Andante involved in the Drosophila circadian oscillator. Mol Cell Biochem 274, 151–161 (2005). https://doi.org/10.1007/s11010-005-2944-0

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-005-2944-0

Keywords

Search

Navigation