Tuning a three-component reaction for trapping kinase substrate complexes

Alexander V. Statsuk, Dustin J. Maly, Markus A. Seeliger, Miles A. Fabian, William H. Biggs, David J. Lockhart, Patrick P. Zarrinkar, John Kuriyan, Kevan M. Shokat

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase- binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.

Original languageEnglish (US)
Pages (from-to)17568-17574
Number of pages7
JournalJournal of the American Chemical Society
Volume130
Issue number51
DOIs
StatePublished - Dec 24 2008

ASJC Scopus subject areas

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

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