title: Identification of the catalytic site of phospholipase D2 (PLD2) newly described guanine nucleotide exchange factor activity.
abstract: We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306-Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Mahankali
authors.forename: Karen M
authors.initials: KM
authors.lastname: Henkels
authors.forename: Gerald
authors.initials: G
authors.lastname: Alter
authors.forename: Julian
authors.initials: J
authors.lastname: Gomez-Cambronero
mesh_terms: D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002465:Cell Movement; D002633:Chemotaxis; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008049:Lipase / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010587:Phagocytosis; D010739:Phospholipase D / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D020744:rac GTP-Binding Proteins / Q000737:chemistry; D000097766:RAC2 GTP-Binding Protein
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D008049:Lipase; D010739:Phospholipase D; D011994:Recombinant Proteins; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C105606:phospholipase D2