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Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.

ABSTRACT: Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods applied in the identification of PP2Cm as a resident mitochondrial protein phosphatase based on sequence analysis and biochemical characterization. We will also provide experimental protocols used to establish the intracellular localization of PP2Cm, to achieve loss and gain function of PP2Cm in cultured cells and intact tissue, and to assess the impact of PP2Cm deficiency on cell death, mitochondria oxidative phosphorylation and permeability transition pore opening.

SUBMITTER: Lu G

PROVIDER: S-EPMC3705213 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.

Lu Gang G Sun Haipeng H Korge Paavo P Koehler Carla M CM Weiss James N JN Wang Yibin Y

Methods in enzymology 20090101

Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods ...[more]

PMID: 19426872

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Project description:In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys over the past decade in phylogenetically diverse bacteria have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes, such as that of Escherichia coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases, which have extensive sequence and structural homology to eukaryotic Ser/Thr protein phosphatase 2C (PP2C) phosphatases. These proteins, called eukaryote-like Ser/Thr phosphatases (eSTPs), have been identified in a number of bacteria but not in E. coli Here, we describe a previously unknown eSTP encoded by an E. coli open reading frame (ORF), yegK, and characterize its biochemical properties, including its kinetics, substrate specificity, and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely related E. coli strains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase that is encoded in the same operon.IMPORTANCE Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacterium Escherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported in E. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

Project description:BackgroundProtein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.ResultsThe specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.ConclusionsMany characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

Dataset Information

Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.

Publications

Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.

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