Phosphoproteome Study of Escherichia coli Devoid of Ser/Thr Kinase YeaG During the Metabolic Shift From Glucose to Malate
Journal article, 2021

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 Delta yeaG, 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 Delta yeaG exhibits a clear phenotype. By metabolic profiling, we discovered that Delta yeaG 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 Delta yeaG 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.

SILAC

metabolic adaptation

phosphoproteome

kinase-substrate relationship

protein kinase

Author

Abida Sultan

Technical University of Denmark (DTU)

Carsten Jers

Technical University of Denmark (DTU)

Tariq A. Ganief

University of Tübingen

Lei Shi

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Meriem Senissar

Technical University of Denmark (DTU)

Julie Bonne Kohler

Technical University of Denmark (DTU)

Boris Macek

University of Tübingen

Ivan Mijakovic

Technical University of Denmark (DTU)

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Frontiers in Microbiology

1664-302X (ISSN)

Vol. 12 657562

Subject Categories

Cell Biology

Biochemistry and Molecular Biology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.3389/fmicb.2021.657562

PubMed

33889145

More information

Latest update

5/12/2021