An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils
| Autor: | Sylvain G. Bourgoin, François Chouinard, Danielle Harbour, Delphine Faugaret, Mohammed-Amine El Azreq |
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| Rok vydání: | 2011 |
| Předmět: |
rho GTP-Binding Proteins
Molecular Sequence Data Vesicular Transport Proteins Phosphatidic Acids Plasma protein binding Biology Biochemistry Amine transport 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Phospholipase D Humans Amino Acid Sequence cdc42 GTP-Binding Protein Protein kinase C 030304 developmental biology Pharmacology 0303 health sciences Vesicle Cell Membrane nutritional and metabolic diseases Phosphatidic acid rac GTP-Binding Proteins Cell biology Protein Transport Cytosol chemistry Liposomes Calcium RhoG 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | Biochemical Pharmacology. 81:144-156 |
| ISSN: | 0006-2952 |
| DOI: | 10.1016/j.bcp.2010.09.014 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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