BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

A Generic Time-resolved Fluorescence Assay for Serine/threonine Kinase Activity: Application to Cdc7/Dbf4

  • Xu, Kui (Roche Research Center, Hoffmann-La Roche Inc.) ;
  • Stern, Alvin S. (Roche Research Center, Hoffmann-La Roche Inc.) ;
  • Levin, Wayne (Roche Research Center, Hoffmann-La Roche Inc.) ;
  • Chua, Anne (Roche Research Center, Hoffmann-La Roche Inc.) ;
  • Vassilev, Lyubomir T. (Roche Research Center, Hoffmann-La Roche Inc.)
  • Received : 2003.03.04
  • Accepted : 2003.03.10
  • Published : 2003.07.31
Download PDF
⟨ Previous Next ⟩

Abstract

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phosphothreonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the $K_m$ for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

Keywords

References

  1. Bader, B., Butt, E., Palmetshoer, A., Walter, U., Jarchau, T. and Drueckes, P. (2001) A cGMP-dependent protein kinase assay for high throughput screening based on time-resolved fluorescence energy transfer. J. Biomol. Screening 6, 255-264. https://doi.org/10.1177/108705710100600407
  2. Cell Signaling Technologies, Catalog No. 9381, Product insert.
  3. Chang, Q. and Tepperman, B. L. (2001) The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity to a rat intestinal epithelial cell line. Am. J. Physiol. Gastrointest. Liver Physiol. 280, 572-583. https://doi.org/10.1152/ajpgi.2001.280.4.G572
  4. Cross T., Griffiths, G., Deacon, E., Sallis, R., Gough, M., Watters, D. and Lord, J. M. (2000) PKC-delta is an apoptotic lamin kinase. Oncogene 19, 2331-3337. https://doi.org/10.1038/sj.onc.1203555
  5. Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105. https://doi.org/10.1042/0264-6021:3510095
  6. Gaarde, W. A., Hunter, T., Brady, H., Murray, B. W. and Goldman, M. E. (1997) Development of nonradioactive, timeresolved fluorescence assay for the measurement of Jun Nterminal kinase activity. J. Biomol. Screening 2, 213-223. https://doi.org/10.1177/108705719700200406
  7. Gschwendt, M., Muller, H. J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G. and Marks, F. (1994) Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93-98. https://doi.org/10.1006/bbrc.1994.1199
  8. Hess, G., Drong, R., Weiland, K., Slightom, J. , Sclafani, R. and Hollingsworth, R. (1998) A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines. Gene 211, 133-140. https://doi.org/10.1016/S0378-1119(98)00094-8
  9. Hunter, T. (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225-236. https://doi.org/10.1016/0092-8674(95)90405-0
  10. Hunter, T. (2000) Signaling-2000 and beyond. Cell 100, 113-127. https://doi.org/10.1016/S0092-8674(00)81688-8
  11. Jiang, W., McDonald, D., Hope, T. and Hunter, T. (1999) Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication. EMBO J. 18, 5703-5713. https://doi.org/10.1093/emboj/18.20.5703
  12. Lei, M. and Tye, B. K. (2001) Initiating DNA synthesis: from recruiting to activating the MCM complex. J. Cell Sci. 114, 1447-1454.
  13. Mallon, R., Feldberg, L. R., Kim, S. C., Collins, K.,Wojciechowicz, D., Hollander, I. and Kovacs, E. (2001) An enzyme-linked immunosorbent assay for Raf/MEK1/MAPK signaling cascade. Anal. Biochem. 294, 48-54. https://doi.org/10.1006/abio.2001.5151
  14. Masai, H. and Arai, K. (2002) Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell Physiol. 190, 287-296. https://doi.org/10.1002/jcp.10070
  15. Masai, H., Matsui, E., You, Z., Ishimi, Y., Tanmai, K. and Arai, K. (2000) Human Cdc7-related kinase complex. J. Biol. Chem. 275, 29042-29052. https://doi.org/10.1074/jbc.M002713200
  16. Song, J. C., Hanson, C. M., Tsai, V., Farokhzad, O. C., Lotz, M.and Matthews, J. B. (2001) Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am. J. Cell Physiol. 281, 649-661.
  17. Stern, A. S. and Podlaski, F. J. (1993) Increasing the antigen binding capacity of immobilized antibodies; in Techniques in Protein Chemistry IV, pp. 353-360, Academic Press Inc., San Diego, USA.
  18. Tabakoff, B., Nelson, E., Yoshimura, M., Hellevuo, K. andHoffman. P. L. (2001) Phosphorylation cascades control the actions of ethanol on cell cAMP signaling. J. Biomed. Sci. 8, 44-51. https://doi.org/10.1007/BF02255970
  19. Todorov, I. T., Pepperkok, R., Philipova, R., Kearsey, S. E.,Ansorge, W. and Werner, D. A. (1994) A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division. J. Cell Sci. 107, 253-265.

Cited by

  1. Alternative assay formats to identify diverse inhibitors of protein kinases vol.3, pp.7, 2008, https://doi.org/10.1517/17460441.3.7.819
  2. Progress in Lanthanides as Luminescent Probes vol.15, pp.4, 2005, https://doi.org/10.1007/s10895-005-2826-6
  3. Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay vol.7, pp.1, 2009, https://doi.org/10.1089/adt.2008.175
  4. Synthesis and evaluation of pyrido-thieno-pyrimidines as potent and selective Cdc7 kinase inhibitors vol.19, pp.2, 2009, https://doi.org/10.1016/j.bmcl.2008.11.093
  5. Transcreener™: screening enzymes involved in covalent regulation vol.10, pp.1, 2006, https://doi.org/10.1517/14728222.10.1.179