Advertisement

Affinity Purification of Protein Kinases that Adopt a Specific Inactive Conformation

  • Pratistha Ranjitkar
  • Dustin J. MalyEmail author
Protocol
  • 2k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 928)

Abstract

Several protein kinases have been characterized in a specific inactive form called the DFG-out conformation. Unlike the active conformation which is conserved in all kinases, the inactive DFG-out conformation appears to be accessible to only certain kinases. This inactive conformation has been successfully targeted with highly selective kinase inhibitors, including the cancer drugs imatinib and sorafenib. However, the structural and sequence requirements for adopting this conformation are still poorly understood. Here, we describe a general method for enriching DFG-out adopting kinases from cell lysates with an affinity resin that contains a general ligand that specifically recognizes this inactive form.

Key words

Protein kinase DFG-out conformation Affinity purification Inhibitors Protein conformation Protein ligands 

Notes

Acknowledgment

This work was supported by the National Institute of General Medical Science (R01GM086858).

References

  1. 1.
    Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298:1912–1934PubMedCrossRefGoogle Scholar
  2. 2.
    Cohen P (2000) The regulation of protein function by multisite phosphorylation–a 25 year update. Trends Biochem Sci 25:596–601PubMedCrossRefGoogle Scholar
  3. 3.
    Cohen P (2002) Protein kinases–the major drug targets of the twenty-first century? Nat Rev Drug Discov 1:309–315PubMedCrossRefGoogle Scholar
  4. 4.
    Liu Y, Gray NS (2006) Rational design of inhibitors that bind to inactive kinase conformations. Nat Chem Biol 2:358–364PubMedCrossRefGoogle Scholar
  5. 5.
    Okram B, Nagle A, Adrian FJ, Lee C, Ren P, Wang X, Sim T, Xie Y, Wang X, Xia G, Spraggon G, Warmuth M, Liu Y, Gray NS (2006) A general strategy for creating “inactive-conformation” abl inhibitors. Chem Biol 13:779–786PubMedCrossRefGoogle Scholar
  6. 6.
    Seeliger MA, Ranjitkar P, Kasap C, Shan Y, Shaw DE, Shah NP, Kuriyan J, Maly DJ (2009) Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations. Cancer Res 69:2384–2392PubMedCrossRefGoogle Scholar
  7. 7.
    Ranjitkar P, Brock AM, Maly DJ, Affinity reagents that target a specific inactive form of protein kinases. Chem. Biol. 17:195–206.Google Scholar
  8. 8.
    Knockaert M, Gray N, Damiens E, Chang YT, Grellier P, Grant K, Fergusson D, Mottram J, Soete M, Dubremetz JF, Le Roch K, Doerig C, Schultz P, Meijer L (2000) Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors. Chem Biol 7:411–422PubMedCrossRefGoogle Scholar
  9. 9.
    Knockaert M, Wieking K, Schmitt S, Leost M, Grant KM, Mottram JC, Kunick C, Meijer L (2002) Intracellular Targets of Paullones. Identification following affinity purification on immobilized inhibitor. J Biol Chem 277:25493–25501PubMedCrossRefGoogle Scholar
  10. 10.
    Wissing J, Jansch L, Nimtz M, Dieterich G, Hornberger R, Keri G, Wehland J, Daub H (2007) Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry. Mol Cell Proteomics 6:537–547PubMedGoogle Scholar
  11. 11.
    Godl K, Wissing J, Kurtenbach A, Habenberger P, Blencke S, Gutbrod H, Salassidis K, Stein-Gerlach M, Missio A, Cotten M, Daub H (2003) An efficient proteomics method to identify the cellular targets of protein kinase inhibitors. Proc Natl Acad Sci USA 100:15434–15439PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA

Personalised recommendations