Development of photo‐crosslinking reagents for protein kinase–substrate interactions

FEBS Letters

Cole, Philip A.¹  Kohn, Jeffrey A.¹  Saldanha, S.Adrian¹  Parang, Keykavous¹ 

[1] Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

关键词: Protein kinase;    Csk;    Src;    Nucleotide;    Photo-crosslinking;   

DOI  :  10.1016/S0014-5793(02)02778-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown