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Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein

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Published/Copyright: November 24, 2005
Biological Chemistry
From the journal Volume 386 Issue 11

Abstract

Lysophosphatidic acid is a pleiotropic lipid signalingmolecule that evokes a broad array of cellular responses including proliferation, tumor cell invasion, neurite retraction, cytoskeletal rearrangements and smooth muscle contraction. Generally, lysophosphatidic acid triggers physiological responses through interaction with specific plasma membrane receptors called LPA 1–4. There is, however, increasing evidence in support of intracellular proteins that interact with LPA. We employed Affigel-immobilized LPA to isolate cytoplasmic proteins that interact with this lysophospholipid. Among the proteins retained by this affinity matrix, pyruvate kinase, clathrin heavy chain and heat shock protein 70 (Hsp70) were identified by mass spectrometry. Isothermal titration calorimetry showed that pyruvate kinase contains onebinding site for LPA (Ka approx. 106 M-1). Furthermore, LPA dissociates enzymatically active pyruvate-kinase tetramers into less active dimers, and is maximally active at concentrations close to its critical micelle concentration. These effects were not mimicked by other lysophospholipids. Co-immunoprecipitation experiments showed that pyruvate kinase interacts with clathrin, and confocal imaging revealed co-localization between clathrin and pyruvate kinase in the perinuclear region of cells. Our data suggest that pyruvate kinase partly exists in complex with clathrin in subcellular membranous areas, and that locally increased LPA levels can trigger inactivation of the metabolic enzyme.

Keywords: circular dichroism; enzymatic activity; gel filtration; isothermal calorimetry titration; mass spectrometry; phospholipid binding
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Published Online: 2005-11-24
Published in Print: 2005-11-01

©2005 by Walter de Gruyter Berlin New York

Articles in the same Issue

  1. Supplementary material to the paper "B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade"
  2. Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface
  3. Macromolecular isoforms of Daphnia magna haemoglobin
  4. Rational engineering of a fluorescein-binding anticalin for improved ligand affinity
  5. Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins
  6. ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria
  7. Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite
  8. Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein
  9. The polysaccharide scaffold of PrP 27-30 is a common compound of natural prions and consists of α-linked polyglucose
  10. Cytosolic persistence of mouse brain CYP1A1 in chronic heme deficiency
  11. B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade
  12. Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)
  13. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI
  14. Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L
  15. Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects
https://doi.org/10.1515/BC.2005.130
Keywords for this article
circular dichroism; enzymatic activity; gel filtration; isothermal calorimetry titration; mass spectrometry; phospholipid binding

Articles in the same Issue

  1. Supplementary material to the paper "B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade"
  2. Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface
  3. Macromolecular isoforms of Daphnia magna haemoglobin
  4. Rational engineering of a fluorescein-binding anticalin for improved ligand affinity
  5. Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins
  6. ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria
  7. Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite
  8. Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein
  9. The polysaccharide scaffold of PrP 27-30 is a common compound of natural prions and consists of α-linked polyglucose
  10. Cytosolic persistence of mouse brain CYP1A1 in chronic heme deficiency
  11. B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade
  12. Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)
  13. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI
  14. Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L
  15. Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects
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