An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils

Biochem Pharmacol. 2011 Jan 1;81(1):144-56. doi: 10.1016/j.bcp.2010.09.014. Epub 2010 Sep 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Calcium / metabolism
  • Cell Membrane / metabolism*
  • Humans
  • Liposomes
  • Molecular Sequence Data
  • Phosphatidic Acids / metabolism
  • Phospholipase D / metabolism*
  • Protein Binding
  • Protein Transport
  • RAC2 GTP-Binding Protein
  • Vesicular Transport Proteins / chemistry
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism*
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism
  • rac GTP-Binding Proteins / genetics
  • rac GTP-Binding Proteins / metabolism
  • rho GTP-Binding Proteins / genetics
  • rho GTP-Binding Proteins / metabolism


  • Liposomes
  • Phosphatidic Acids
  • VAT1 protein, human
  • Vesicular Transport Proteins
  • RHOG protein, human
  • Phospholipase D
  • cdc42 GTP-Binding Protein
  • rac GTP-Binding Proteins
  • rho GTP-Binding Proteins
  • Calcium