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Development of photo‐crosslinking reagents for protein kinase–substrate interactions
Author(s) -
Parang Keykavous,
Kohn Jeffrey A.,
Saldanha S.Adrian,
Cole Philip A.
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02778-3
Subject(s) - protein kinase a , biochemistry , chemistry , substrate (aquarium) , kinase , ternary complex , protein kinase c , enzyme , biophysics , combinatorial chemistry , biology , ecology
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.