title: Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2.
abstract: We have discovered that the enzyme phospholipase D2 (PLD2) binds directly to the small GTPase Rac2, resulting in PLD2 functioning as a guanine nucleotide exchange factor (GEF), because it switches Rac2 from the GDP-bound to the GTP-bound states. This effect is large enough to be meaningful (∼72% decrease for GDP dissociation and 300% increase for GTP association, both with PLD2), it has a half-time of ∼7 min, is enhanced with increasing PLD2 concentrations, and compares favorably with other known GEFs, such as Vav-1. The PLD2-Rac2 protein-protein interaction is sufficient for the GEF function, because it can be demonstrated in vitro with just recombinant proteins without lipid substrates, and a catalytically inactive lipase (PLD2-K758R) has GEF activity. Apart from this function, exogenous phosphatidic acid by itself (300 pM) increases GTP binding and enhances PLD2-K758R-mediated GTP binding (by ∼34%) but not GDP dissociation. Regarding the PLD2-Rac2 protein-protein association, it involves, for PLD2, residues 263-266 within a Cdc42/Rac interactive binding region in the PH domain, as well as the PX domain, and it involves, for Rac2, residue N17 within its Switch-1 region. PLD2's GEF function is demonstrated in living cells, because silencing PLD2 results in reduced Rac2 activity, whereas PLD2-initiated Rac2 activation enhances cell adhesion, chemotaxis, and phagocytosis. There are several known GEFs, but we report that this GEF is harbored in a phospholipase. The benefit to the cell is that PLD2 brings spatially separated molecules together in a membrane environment, ready for fast intracellular signaling and cell function.
authors: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
authors: Madhu
authors: M
authors: Mahankali
authors: Hong-Juan
authors: HJ
authors: Peng
authors: Karen M
authors: KM
authors: Henkels
authors: Mary C
authors: MC
authors: Dinauer
authors: Julian
authors: J
authors: Gomez-Cambronero
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D009154:Mutation; D010712:Phosphatidic Acids / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010712:Phosphatidic Acids; D010739:Phospholipase D; D011994:Recombinant Proteins; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C105606:phospholipase D2