Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein.
Lysophosphatidic acid is a pleiotropic lipid signaling molecule 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 one binding site for LPA (Ka approx. 10(6) 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.
Desmaret
Sophie
S
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Faculty of Medicine and Health Sciences, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
Qian
Lian
L
Vanloo
Berlinda
B
Meerschaert
Kris
K
Van Damme
Jozef
J
Grooten
Johan
J
Vandekerckhove
Joël
J
Prestwich
Glenn D
GD
Gettemans
Jan
J
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D008246:Lysophospholipids / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits
D002352:Carrier Proteins; D002966:Clathrin; D018840:HSP70 Heat-Shock Proteins; D008246:Lysophospholipids; D046912:Multiprotein Complexes; D011770:Pyruvate Kinase; C032881:lysophosphatidic acid