Advertisement

Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening

  • Clint D. J. Tavares
  • Ashwini K. Devkota
  • Kevin N. Dalby
  • Eun Jeong Cho
Part of the Methods in Molecular Biology book series (MIMB, volume 1360)

Abstract

Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.

Key words

Kinase eEF-2K Expression Purification Fluorescence Luminescence Radioactive High-throughput screen 

Notes

Acknowledgements

This research was supported in part by the grants from the Welch Foundation (F-1390), CPRIT (RP110532-P1), and NIH (GM059802 and CA167505).

References

  1. 1.
    Wu H, Yang JM, Jin S et al (2006) Elongation factor-2 kinase regulates autophagy in human glioblastoma cells. Cancer Res 66:3015–3023CrossRefPubMedGoogle Scholar
  2. 2.
    Hait WN, Wu H, Jin S et al (2006) Elongation factor-2 kinase: its role in protein synthesis and autophagy. Autophagy 2:294–296CrossRefPubMedGoogle Scholar
  3. 3.
    Calberg U, Nilsson A, Skog S et al (1991) Increased activity of the eEF-2 specific, Ca2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells. Biochem Biophys Res Commun 180:1372–1376CrossRefPubMedGoogle Scholar
  4. 4.
    Parmer TG, Ward MD, Yurkow EJ et al (1999) Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. Br J Cancer 79:59–64PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Bagaglio DM, Cheng EH, Gorelick FS et al (1993) Phosphorylation of elongation factor 2 in normal and malignant rat glial cells. Cancer Res 53:2260–2264PubMedGoogle Scholar
  6. 6.
    Hait WN, Ward MD, Trakht IN et al (1996) Elongation factor-2 kinase: immunological evidence for the existence of tissue-specific isoforms. FEBS Lett 397:55–60CrossRefPubMedGoogle Scholar
  7. 7.
    Parmer TG, Ward MD, Hait WN (1997) Effects of rottlerin, an inhibitor of calmodulin-dependent protein kinase III, on cellular proliferation, viability, and cell cycle distribution in malignant glioma cells. Cell Growth Differ 8:327–334PubMedGoogle Scholar
  8. 8.
    Cheng Y, Li H, Ren X et al (2010) Cytoprotective effect of the elongation factor-2 kinase-mediated autophagy in breast cancer cells subjected to growth factor inhibition. PLoS One 5, e9715PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Wu H, Zhu H, Liu DX et al (2009) Silencing of elongation factor-2 kinase potentiates the effect of 2-deoxy-D-glucose against human glioma cells through blunting of autophagy. Cancer Res 69:2453–2460PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Tekedereli I, Alpay SN, Tavares CD et al (2012) Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer. PLoS One 7, e41171PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Autry AE, Adachi M, Nosyreva E et al (2011) NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475:91–95PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Leprivier G, Remke M, Rotblat B et al (2013) The eEF2 Kinase Confers Resistance to Nutrient Deprivation by Blocking Translation Elongation. Cell 153:1064–1079PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Arora S, Yang JM, Kinzy TG et al (2003) Identification and characterization of an inhibitor of eukaryotic elongation factor 2 kinase against human cancer cell lines. Cancer Res 63:6894–6899PubMedGoogle Scholar
  14. 14.
    Devkota AK, Tavares CD, Warthaka M et al (2012) Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: evidence of a common in vitro artifact. Biochemistry 51:2100–2112PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Chen Z, Gopalakrishnan SM, Bui MH et al (2011) 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor. J Biol Chem 286:43951–43958PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Parker MW, Lo BM, Federici G (1990) Crystallization of glutathione S-transferase from human placenta. J Mol Biol 213:221–222CrossRefPubMedGoogle Scholar
  17. 17.
  18. 18.
    von Ahsen O, Bomer U (2005) High-throughput screening for kinase inhibitors. Chembiochem 6:481–490CrossRefGoogle Scholar
  19. 19.
    Shults MD, Imperiali B (2003) Versatile fluorescence probes of protein kinase activity. J Am Chem Soc 125:14248–14249CrossRefPubMedGoogle Scholar
  20. 20.
    Shults MD, Janes KA, Lauffenburger DA et al (2005) A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates. Nat Methods 2:277–283CrossRefPubMedGoogle Scholar
  21. 21.
  22. 22.
    Tavares CD, O'Brien JP, Abramczyk O et al (2012) Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence. Biochemistry 51:2232–2245PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Devkota AK, Warthaka M, Edupuganti R et al (2014) High-Throughput Screens for eEF-2 Kinase. J Biomol Screen 19:445–452CrossRefPubMedGoogle Scholar
  24. 24.
    Kapust RB, Tozser J, Fox JD et al (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14:993–1000CrossRefPubMedGoogle Scholar
  25. 25.
    Kristelly R, Earnest BT, Krishnamoorthy L et al (2003) Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF. Acta Crystallographica Sect D 59:1859–1862CrossRefGoogle Scholar
  26. 26.
    Gaertner TR, Kolodziej SJ, Wang D et al (2004) Comparative Analyses of the Three-dimensional Structures and Enzymatic Properties of α, β, γ, and δ Isoforms of Ca2+-Calmodulin-dependent Protein Kinase II. J Biol Chem 279:12484–12494CrossRefPubMedGoogle Scholar
  27. 27.
    Forest A, Swulius MT, Tse JKY et al (2008) Role of the N- and C-Lobes of Calmodulin in the Activation of Ca2+/Calmodulin-Dependent Protein Kinase II. Biochemistry 47:10587–10599PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Abramczyk O, Tavares CDJ, Devkota AK et al (2011) Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli. Protein Expr Purif 79:237–244PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Zhang JH, Chung TD, Oldenburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4:67–73CrossRefPubMedGoogle Scholar
  30. 30.
    Taylor JA, Burton NP, Maxwell A (2012) High-throughput microtitre plate-based assay for DNA topoisomerases. Methods Mol Biol 815:229–239CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Medicinal Chemistry, College of PharmacyThe University of Texas at AustinAustinUSA
  2. 2.Texas Screening Alliance for Cancer TherapeuticsThe University of Texas at AustinAustinUSA
  3. 3.College of PharmacyThe University of Texas at AustinAustinUSA

Personalised recommendations