An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils.
Although phosphatidic acid (PA) regulates a wide variety of physiological processes, its targets remain poorly characterized in human neutrophils. By co-sedimentation with PA-containing vesicles we identified several PA-binding proteins including vesicle amine transport protein-1 (VAT-1), Annexin A3 (ANXA3), Rac2, Cdc42 and RhoG in neutrophil cytosol. Except for ANXA3, protein binding to PA-containing liposomes was calcium-independent. Cdc42 and RhoG preferentially interacted with PA whereas VAT-1 bound to PA or phosphatidylserine with the same affinity. VAT-1 translocated to neutrophil membranes upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation. Inhibition of fMLF-induced PLD activity with the Src kinase inhibitor PP2, the selective inhibitor of PLD FIPI, or of PA formation with primary alcohols reduced VAT-1 translocation. In contrast, inhibition of PA hydrolysis with propranolol enhanced fMLF-mediated VAT-1 recruitment to membranes. PMA also redistributed VAT-1 to membranes in a PKC- and PLD-dependent manner. Though fMLF and PMA increased VAT-1 phosphorylation, different kinases appear to be involved. Cell fractionation revealed that a pool of VAT-1 was co-localized with primary, secondary and tertiary granules and plasma membrane markers in resting neutrophils. Stimulation with fMLF enhanced VAT-1 co-localization with CD32a, a plasma membrane marker. Confocal microscopy revealed that VAT-1 decorates granular structures at the cell periphery and double labeling with VAT-1/lactoferrin antibodies showed a partial co-localization with secondary granules in control and fMLF-stimulated cells. Characterization of these putative PA-binding proteins constitutes another step forward for a better understanding of the role of PLD-derived PA in neutrophil physiology.
Faugaret
Delphine
D
Centre de Recherche en Rhumatologie et Immunologie, Centre de recherche du CHUQ-CHUL et Faculté de Médecine de l'Université Laval, 2705 Boulevard Laurier, local T1-49, Québec, QC, G1V 4G2, Canada. delphine.faugaret@crchul.ulaval.ca
Chouinard
François C
FC
Harbour
Danielle
D
El azreq
Mohammed-Amine
MA
Bourgoin
Sylvain G
SG
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
D002118:Calcium; D008081:Liposomes; D010712:Phosphatidic Acids; D010739:Phospholipase D; D033921:Vesicular Transport Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C544158:VAT1 protein, human; C075397:RHOG protein, human