title: The dual effect of Rac2 on phospholipase D2 regulation that explains both the onset and termination of chemotaxis.
abstract: We document a biphasic effect of Rac2 on the activation and inhibition of PLD2. Cells overexpressing Rac2 and PLD2 simultaneously show a robust initial (<10 min) response toward a chemoattractant that is later (>30 min) greatly diminished over PLD2-only controls. The first phase is due to the presence of a Rac2-PLD2 positive-feedback loop. To explain the mechanism for the Rac2-led PLD2 inhibition (the second phase), we used leukocytes from wild-type (WT) and Rac2(-/-) knockout mice. Rac2(-/-) cells displayed an enhanced PLD2 (but not PLD1) enzymatic activity, confirming the inhibitory role of Rac2. Late inhibitory responses on PLD2 due to Rac2 were reversed in the presence of phosphatidylinositol 4,5-bisphosphate (PIP(2)) both in vitro (purified GST-PH-PLD2, where GST is glutathione S-transferase and PH is pleckstrin homology) and in vivo. Coimmunoprecipitation and immunofluorescence microscopy indicated that PLD2 and Rac2 remain together. The presence of an "arc" of Rac2 at the leading edge of leukocyte pseudopodia and PLD2 physically posterior to this wave of Rac2 was observed in late chemotaxis. We propose Rac-led inhibition of PLD2 function is due to sterical interference of Rac with PLD2's PH binding site to the membrane and deprivation of the PIP(2). This work supports the importance of functional interactions between PLD and Rac in the biological response of cell migration.
authors: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
authors: Hong-Juan
authors: HJ
authors: Peng
authors: Karen M
authors: KM
authors: Henkels
authors: Madhu
authors: M
authors: Mahankali
authors: Christophe
authors: C
authors: Marchal
authors: Paula
authors: P
authors: Bubulya
authors: Mary C
authors: MC
authors: Dinauer
authors: Julian
authors: J
authors: Gomez-Cambronero
mesh_terms: D000818:Animals; D001798:Blood Proteins; D002633:Chemotaxis; D002634:Chemotaxis, Leukocyte; D005982:Glutathione Transferase / Q000378:metabolism; D047468:Immunoprecipitation; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010739:Phospholipase D / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D011485:Protein Binding; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D001798:Blood Proteins; D005982:Glutathione Transferase; D019269:Phosphatidylinositol 4,5-Diphosphate; D010739:Phospholipase D; D010750:Phosphoproteins; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C039077:platelet protein P47; C105606:phospholipase D2