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Sophie Desmaret, Lian Qian, Berlinda Vanloo, Kris Meerschaert, Jozef Van Damme, Johan Grooten, Joël Vandekerckhove, Glenn D. Prestwich, Jan Gettemans

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

Keywords: circular dichroism, enzymatic activity, gel filtration, isothermal calorimetry titration, Mass spectrometry, phospholipid binding

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 (K_a 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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 386, 11/2005
Pages: 1137 - 1147

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