Chemical tools to study protein arginine phosphorylation

Post-translation modifications of proteins by reversible phosphorylation represent a crucial hallmark of all organisms. This type of modification is dynamically regulated by the activity of protein kinases that transfer phospho-groups onto the side chain of diverse amino-acid residues and by protein phosphatases that efficiently remove the phospho-moieties. Protein phosphorylation is involved in numerous cellular signaling events and its dysregulation is implicated in a vast variety of different diseases including cancer, thereby explaining its central importance to both, academic and biopharmaceutical research. Protein kinases typically phosphorylate the side chains of serine, threonine and tyrosine residues. However, more recently, the bacterial McsB protein was unequivocally identified as the first protein arginine kinase. Subsequent work identified the YwlE enzyme as its cognate arginine phosphatase that efficiently hydrolyzes phosphoarginine residues in vitro and in vivo. The McsB and YwlE arginine kinase/phosphatase couple controls the reversible arginine phosphorylation of target proteins such as the bacterial stress response regulator CtsR. In addition, it was shown that protein arginine phosphorylation affects a variety of cellular pathways including the stress response, protein degradation, cell motility, and competence development in Bacillus subtilis. Although the impact of protein arginine phosphorylation is beginning to come into focus, much remains to be learned and new chemical tools to selectively enrich and/or detect the enzymes responsible for these modifications are needed to accelerate research in this emerging field. The proposed project aims at generating chemical probes to target protein arginine phosphatases. The envisaged probes consist of a tripartite component encompassing a central active site directed phosphoarginine mimicking moiety that is linked via a photo-reactive group to a reporter tag. We are planning to use these probes to visualize and enrich protein arginine phosphatases and/or phosphoarginine binding proteins. The probes will be initially evaluated using recombinant YwlE, followed by cellular studies in Bacillus subtilis. To identify novel protein arginine phosphatases, we will utilize these photo-probes to analyse distinct bacterial organisms, as well as mammalian cell lines. To test the potential phosphoarginine phosphatase activity of the identified phosphatase enzymes, we will also synthesize a novel phosphoarginine substrate that can be used for rapid screening assays. The project ultimately aims to design and synthesize chemical tools to probe, identify and evaluate known and novel phosphoarginine phosphatases thereby providing new insights about the cellular regulation and the prevalence of protein arginine phosphorylation. In this regard, the results of the proposed work will greatly facilitate the investigation of protein arginine phosphorylation in diverse model organisms.

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