Phosphoproteome Study of Escherichia coli Devoid of Ser/Thr Kinase YeaG During the Metabolic Shift From Glucose to Malate

Abstract

Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

“…3d). The site occupancy of S177 and T179 was recently reported to be around 15% during growth on glucose 13 , a percentage that significantly decreases TpiA in vitro activity (Supplementary Fig. 1) and hence glycolytic flux.…”

“…Thus, phosphoregulation may also be prevalent in bacteria, although only a few S/T-and Y-kinases and phosphatases are known and their in vivo substrates and regulators remain poorly characterized 12,13 . Mere detection of a phosphosite provides little evidence for function 14,15 , which is typically inferred indirectly from phosphosite conservation, co-occurrence with other modifications, and correlation of the degree of protein phosphorylation with physiological variables such as metabolic flux [16][17][18][19][20][21] .…”

Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli

“…[6][7][8][9][10][11][12][13] Similar to eukaryotes, the genome of E. coli contains kinases and acetyltransferases that enzymatically catalyze protein phosphorylation and acetylation, respectively, but the knowledge about their targets is limited. 10,14,15 Additionally, proteins can be non-enzymatically modified by reactive metabolites, and some of these chemical modifications can be reversed, providing an additional layer of regulation to cellular processes. 16 The highenergy metabolite acetyl phosphate (AcP) can non-enzymatically acetylate proteins, 17 and its intracellular abundance has been linked to the acetylation of at least a third of all detected acetylation sites in E. coli.…”

PrkA is an ATP-dependent protease that regulates sporulation in Bacillus subtilis

“…In this work, we showed that, despite distant homology with (cAMP)-dependent protein kinases for its C-terminal domain (12) and with the Ser/Thr kinase YeaG from E. coli (13)(14)(15), no autophosphorylation or kinase activity was detected for PrkA whatever the conditions tested. In addition, our data did not highlight an ATP binding site in the C terminal domain of PrkA.…”

“…Furthermore, no PrkA autophosphorylation was detected which was surprising since STPKs generally need to be autophosphorylated to be active. Recently, a homologous protein from Escherichia coli, YeaG, has been shown to be indeed a kinase that autophosphorylates (14) and phosphorylates AceA, the isocitrate lyase involved in the glyoxylic cycle, in the presence of malate (15), but this catalytic pathway is missing in B. subtilis.…”