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.
Highlights
Phospholipase D2 harbors a newly described guanine nucleotide exchange factor (GEF) activity
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
This study demonstrated that recombinant PLD2 itself is a novel class of GEF that lacks the DH-PH domain but possesses the a PX-PH domain
Summary
Phospholipase D2 harbors a newly described GEF activity. Results: The domains and residues responsible for the GEF activity of PLD2 and their physiological relevance in vivo have been identified. We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Catalytic Site of PLD2 Newly Described GEF Activity conventional DH domain in tandem with a PH domain, we hypothesized and proved that the PLD2 PX domain could serve in the role of and function as that of the DH domain in conventional GEFs. Following methodical mutational analysis, we successfully determined that the residues phenylalanines 107 and 129, arginine 172, and leucines 166 and 173 in the PLD2 PX domain are the key amino acid residues responsible for its guanine nucleotide exchange reaction. Ascertaining the exact amino acids that encompass the catalytic activity will be crucial for future rational design of pharmaceuticals to down-regulate leukocytes in situations where they can be harmful to human tissues
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