5‘-(p-Fluorosulfonylbenzoyl)-2‘(or 3‘)-(methylanthraniloyl)adenosine, Fluorescent Affinity Labels for Adenine Nucleotide Binding Sites:  Interaction with the Kinase Active Site of the Receptor for Epidermal Growth Factor

Abstract

We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the kinase domain of the EGF receptor, we prepared new derivatives of 5'-FSBAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (FSBMantAdo), as fluorescent affinity labels for adenine nucleotide binding sites, and in particular for the ATP site of the EGF receptor. The two products were purified by HPLC and were characterized by UV-Vis absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy. Incubation of membrane vesicles containing the EGF receptor with either the 2' or 3' derivative resulted in irreversible inhibition of the receptor kinase activity, as assessed by autophosphorylation assays. Preincubation of vesicles with AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the receptor kinase activity' from FSBMantAdo inactivation. Steady state fluorescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled receptor (426 nm), as compared with the emission maximum of free FSBMantAdo (441 nm) in aqueous solution, suggesting that the receptor-bound label is in a relatively low polarity environment. These studies show that FSBMantAdo is a specific affinity label for the ATP site of the EGF receptor. FSBMantAdo may also prove useful as a fluorescent affinity label for other ATP binding sites.

Kinetic mechanism and ATP‐binding site reactivity of p38γ MAP kinase

“…FSBA‐modified protein was digested with Lys‐C protease and the reversed phase HPLC peptide map compared with that of unmodified p38γ. The overlaid digest maps for p38γ and FSBA‐modified p38γ appeared identical except for the shifted retention time of one peptide that exhibited an absorbance peak centered at 245 nm, characteristic of FSBA derivatives [24]. No other peptide peaks showed this absorbance profile.…”

“…FSBA is an affinity label for nucleotide binding sites that, unlike the non‐hydrolyzable AMP‐PCP, binds irreversibly to the sidechain of a critical, conserved Lys residue found in the ATP‐binding site of most kinases [24]. FSBA is similar in structure to ATP (Fig.…”

Synthesis and Characterization of 5′-p-fluorosulfonylbenzoyl-2′ (or 3′)-(biotinyl)adenosine as an Activity-Based Probe for Protein Kinases

“…Based on the studies of Scoggins et al, 20 we synthesized 2´-biotinyl-FSBA and 3´-biotinyl-FSBA where biotin was attached via its carboxylic acid to the 2´-or 3´-hydroxyl moiety of the ribose group of FSBA. The ATP and staurosporine protection studies confirmed that the mode of labeling of biotinyl-FSBA to protein kinases was identical to that of FSBA.…”

“…18 FSBA covalently modifies a conserved lysine present in the nucleotide binding sites of most protein kinases. [18][19][20][21] To generate biotin-tagged FSBA, we built on the earlier work of Scoggins et al 20 by modifying the free hydroxyl groups of the ribose ring of FSBA with biotin (Fig. 1).…”

Synthesis and Evaluation of Activity‐Based Probes Carrying a 5′‐Fluorosulfonylbenzoyl Adenosine Moiety for Protein Kinases

“…5'-FSBA and its derivatives were also shown to react mainly with the conserved lysine residue in the ATP binding pocket. 16,17 Recently, probes derived from FSBA with a biotin reporter group were reported (probes 1a and 1b). 18 The labeling performance of probes 1a and 1b was roughly the same in the model study using ALK5 and CDK2.…”