Development of photo-crosslinking reagents for protein kinase-substrate interactions

Keykavous Parang, Jeffrey A. Kohn, S. A. Saldanha, Philip A. Cole

Research output: Contribution to journalArticlepeer-review

Abstract

The identification of relevant protein kinase-protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the γ-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein-protein crosslinking that is dependent on UV irradiation. That significant kinase-substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.

Original languageEnglish (US)
Pages (from-to)156-160
Number of pages5
JournalFEBS Letters
Volume520
Issue number1-3
DOIs
StatePublished - Jun 5 2002

Keywords

  • Csk
  • Nucleotide
  • Photo-crosslinking
  • Protein kinase
  • Src

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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