A coiled-coil enabled split-luciferase three-hybrid system: Applied toward profiling inhibitors of protein kinases

Benjamin W. Jester, Kurt J. Cox, Alicia Gaj, Carolyn D. Shomin, Jason R. Porter, Indraneel Ghosh

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades, there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil-assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors.

Original languageEnglish (US)
Pages (from-to)11727-11735
Number of pages9
JournalJournal of the American Chemical Society
Volume132
Issue number33
DOIs
StatePublished - Aug 25 2010

Fingerprint

Protein Kinase Inhibitors
Luciferases
Hybrid systems
Protein Kinases
Proteins
Staurosporine
Phosphotransferases
Firefly Luciferases
Signal transduction
Human Genome
Libraries
Assays
Signal Transduction
Screening
Genes
Enzymes
Testing

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

A coiled-coil enabled split-luciferase three-hybrid system : Applied toward profiling inhibitors of protein kinases. / Jester, Benjamin W.; Cox, Kurt J.; Gaj, Alicia; Shomin, Carolyn D.; Porter, Jason R.; Ghosh, Indraneel.

In: Journal of the American Chemical Society, Vol. 132, No. 33, 25.08.2010, p. 11727-11735.

Research output: Contribution to journalArticle

Jester, Benjamin W. ; Cox, Kurt J. ; Gaj, Alicia ; Shomin, Carolyn D. ; Porter, Jason R. ; Ghosh, Indraneel. / A coiled-coil enabled split-luciferase three-hybrid system : Applied toward profiling inhibitors of protein kinases. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 33. pp. 11727-11735.
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