pArg proteome

Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. It is thus not surprising that also the phosphorylation of arginine residues is under control of a specific kinase-phosphatase couple, the McsB kinase and the YwlE phosphatase. Like its cognate enzymes, the pArg modification itself displays properties that make it unique in the phosphorylation field. Most importantly, arginine phosphorylation results in a positive-to-negative charge inversion. Because arginine residues are frequently observed at molecular interfaces mediating protein-DNA, protein-RNA or protein-protein interactions, the phosphorylation of arginine residues has a huge potential to serve as a molecular switch regulating protein function. Moreover, the phosphate is attached through a phosphoramidate bond. This N-P linkage has chemical properties that are markedly different from those of the O-P phosphoester bond found in pSer, pThr, and pTyr modifications; for example,  it is highly labile under acidic conditions. Such chemical differences may translate into dissimilarity in biological function, but also, from a practical point of view, mean that methods developed to monitor O-P phosphorylations are often not suited to analyze pArg-modified proteins. Therefore, we set out to develop pArg-suited methodologies to characterize the pArg proteome and its impact on cellular signaling.

Our findings: 2014a pArg proteome of B. subtilis, 2014b Development of a pArg trap

2014 Establishing a methodology for pArg proteome analysis

Localization of arginine phosphorylation sites in the ClpC ATPase/unfoldase, the main target of McsB. As most of the identified arginines are not accessible in the hexamer (orange residues) but in the monomer, we presume that the monomer is also the preferentially targeted substrate of McsB.

Although the McsB kinase has been shown to play a crucial role in the stress response of Gram-positive bacteria, its exact in vivo substrates had not been previously identified. Using the recombinant McsB arginine kinase, we could produce arginine phosphorylated protein samples in vitro. This put us in a privileged position to develop mass-spectrometry protocols compatible with the acid-labile properties of phosphoarginine, allowing us to study this modification in vivo in a comprehensive manner. We used the developed phosphoproteomic methods to analyse arginine phosphorylation in a B. subtilis strain lacking the arginine phosphatase YwlE. To our surprise, the McsB kinase phosphorylates a lot of proteins besides its well described target CtsR, one of the master transcriptional regulators of stress response. In fact, we could identify as many as 217 phosphoarginine sites distributed in 134 proteins. Furthermore, quantitative analysis showed that under oxidative and high temperature stress conditions some of these proteins have increased levels of arginine phosphorylation. This indicates that, despite the apparent promiscuity, under stress conditions McsB is specifically targeting a group of substrates – which include major cellular chaperones and proteases as well as the HrcA transcriptional repressor, another master regulator of heat shock gene expression. This study shows that arginine phosphorylation may obtain a much more central role in bacterial physiology than we originally expected, as it may coordinate the bacterial stress response and protein quality control. It will be interesting to see of how the McsB protein arginine kinase fulfill this demanding task on a molecular level.

2014 Development of a pArg trap for proteomics analysis

Selective trapping of pArg (red) proteins by a teethless arginine phosphatase, YwlE.

Even though phosphoarginine has been shown to play a pivotal role in Gram-positive bacteria, the prevalence of arginine phosphorylation in nature remains unknown. However, investigating the presence of phosphoarginine in higher organisms is not an easy task: Ser/Thr protein phosphorylations are extremely abundant in eukaryotic cells and, as a consequence, masks the presence of other less represented phosphorylations during phosphoproteomic analysis. We therefore took advantage of the very specific arginine phosphatase YwlE to develop an affinity tool to isolate arginine-phosphorylated proteins from cell extracts, facilitating their identification. For this purpose, we developed a substrate-trapping mutant of the YwlE phosphatase that retains binding affinity towards arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. By testing a number of active site substitutions, we identified a YwlE mutant (C9A) that stably binds to arginine-phosphorylated proteins. We further improved the substrate-trapping efficiency by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captured arginine-phosphorylated proteins from complex B. subtilis ?ywlE cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications. In conclusion, we present a novel tool for the selective enrichment and subsequent MS analysis of arginine phosphorylation, which is a largely overlooked protein modification that may have an important impact for eukaryotic cell signaling.