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Chemical Tools to Study the Biology of Protein Arginine Phosphorylation
Author(s) -
Fuhrmann Jakob,
Subramanian Venkataraman,
Thompson Paul
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb212
Subject(s) - phosphorylation , amidine , serine , biochemistry , arginine , protein phosphorylation , chemistry , kinase , threonine , protein tyrosine phosphatase , protein kinase a , phosphoserine , tyrosine phosphorylation , biology , amino acid , stereochemistry
Reversible protein phosphorylation is a central mechanism of cellular signaling in both eukaryotes and prokaryotes. The major phosphoacceptor sites comprise serine, threonine and tyrosine residues. Recently, however, phosphorylation of arginine residues was shown to occur in bacterial proteins under stress conditions. There, the protein arginine kinase McsB mediates arginine phosphorylation, while the protein phosphatase YwlE efficiently hydrolyses the generated phosphoramidate (P‐N) bond. The aim of the current study was to generate chemical tools to better understand the physiological regulation of protein arginine phosphorylation. To evaluate the cellular regulation of YwlE we designed and synthesized a diverse set of non‐hydrolyzable phosphoarginine mimetics. The most potent inhibitors contain a sulfonate‐amidine (SO 3 ‐amidine) group and inhibit YwlE with a K i of ~390 nM. We adapted these inhibitors to synthesize SO 3 ‐amidine based photo‐probes. Biochemical and mass spectrometric analyses revealed that these probes specifically recognize the active (reduced) form of YwlE, whereas they do not label the inactive (oxidized) form. Given the high specificity for the active enzyme we utilized this probe to identify the redox regulation of cellular YwlE and thereby derive a mechanistic model of the regulation of arginine phosphorylation under oxidative stress conditions. These findings will stimulate further investigations to characterize the function and regulation of protein arginine phosphorylation under different stress conditions.