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.

Keywords: SILAC; kinase-substrate relationship; metabolic adaptation; phosphoproteome; protein kinase.

Figure 1

Growth phenotype of ΔyeaG during glucose to malate shift. (A) Growth profiler experiment for different Escherichia coli strains (names indicated in the figure inset) shifted from M9 medium supplemented with glucose to fresh M9 medium supplemented with glucose: control with no metabolic shift (average of biological triplicates). (B) Growth profiler experiment for different E. coli strains (names indicated in the figure inset) shifted from M9 medium supplemented with glucose to fresh M9 medium supplemented with malate: metabolic shift from glucose to malate (average of biological triplicates). (C) Growth profile of ΔyeaG and wild type strains in batch cultures scaled up for the stable isotope labeling by amino acids in cell culture (SILAC) proteomics experiment (average of biological duplicates). Time points when samples were collected for the MS proteomics analysis (at OD₆₀₀ = 0.6) are indicated with red arrows.

Figure 2

Differential expression of proteins during glucose to malate shift. (A) Plot of quantified protein ratios (in response to shift in carbon source from glucose to malate) for the ΔyeaG vs. the wild type strain. As a cutoff for assigning proteins differentially regulated in the two strains, we chose to use two SDs from the mean of the dataset (indicated by dotted lines). Proteins that were differentially upregulated or downregulated are marked in black and gene names are shown. (B) Proteins that are differently regulated during malate shift in the wild type and ΔyeaG strains mapped on the central carbon metabolism of E. coli K-12 MG1655. Red arrows are used to highlight metabolic and transport reactions catalyzed by the affected enzymes. The expression change during glucose to malate shift in ΔyeaG compared to the wild type strain is indicated next to the enzyme name by a plus/minus sign.

Figure 3

Putative substrates of YeaG that are potentially involved in the ΔyeaG phenotyope during glucose to malate shift. (A) Proteins that are either less phosphorylated or completely dephosphorylated in ΔyeaG strain during glucose to malate shift, mapped on the central carbon metabolism of E. coli K-12 MG1655. Red arrows signify phosphorylation events differentially downregulated in ΔyeaG, while black arrows indicate phosphorylation events detected only in the wild type strain and undetected in ΔyeaG. (B) Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot images of proteins purified from E. coli. Purified N-terminally His-tagged proteins were probed with anti-phospho-serine and anti-phospho-threonine antibodies, according the phosphorylated residue identified in the SILAC experiment. Positive Western signals are highlighted with red squares. Sizes of examined proteins are: PpsA 87.4 kDa, AceA 48.9 kDA, AcnB 93.5 kDa, SodB 21.3 kDa, and Eno (45.7 kDa). AceA, PpsA, SodB, and Eno were probed with the anti-phospho-threonine antibody, while AcnB was probed with the anti-phospho-serine antibody. Eno was used as a negative control. (C) AceA is phosphorylated by YeaG in vitro. In vitro phosphorylation assay was performed with 6 μg of YeaG (74.5 kDa) and 5 μg of AceA (48.9 kDa), in a 20 μl reaction containing 50 mM HEPES pH 7.4, 10 mM MgCl₂, 10 mM MnCl₂, 100 mM KCl, 10 mM ATP, 0.1% triton-100, and 10 mM malate, and incubated at 37°C for 2 h. Presence of key components (YeaG, AceA, and malate) in the reactions is indicated with +/− above each lane. After the reaction, proteins were separated by SDS-PAGE, with and without Phos-tag (Fujifilm). Bands corresponding to AceA non-phosphorylated and phosphorylated forms are indicated by red arrows.