An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils
An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils
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DOI：
10.1016/j.bcp.2010.09.014
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发表时间：
2011-01-01
影响因子：
5.8
通讯作者：
Bourgoin, Sylvain G.
中科院分区：
文献类型：
--
作者：
Faugaret, Delphine;Chouinard, Francois C.;Bourgoin, Sylvain G.
尽管磷脂酸(PA)调节多种生理过程，但其靶点在人类中性粒细胞中的特性仍然很差。通过与含有PA的小泡共沉淀，我们在中性粒细胞胞浆中鉴定出几种PA结合蛋白，包括VAT-1、Annexin A3(ANXA3)、rac2、CDC42和RhoG。除ANXA3外，蛋白与含PA脂质体的结合不依赖于钙。Cdc42和RhoG优先与PA结合，而VAT-1以相同的亲和力结合PA或磷脂酰丝氨酸。在N-甲酰-甲硫基-亮氨酰苯丙氨酸(FMLF)刺激下，VAT-1转位到中性粒细胞膜上。用Src激酶抑制剂PP2抑制FMLF诱导的PLD活性，PP2是PLD FIPI的选择性抑制剂，或用伯醇抑制PA的形成减少VAT-1的转位。相反，心得安抑制PA的水解可增加FMLF介导的VAT-1在膜上的募集。PMA还以PKC和PLD依赖的方式将VAT-1重新分配到膜上。虽然FMLF和PMA增加了VAT-1的磷酸化，但似乎涉及不同的激酶。细胞分级显示，在静息的中性粒细胞中，VAT-1与初级、次级和三级颗粒以及质膜标志物共定位。FMLF刺激可增强VAT-1与质膜标记物CD32a的共定位。共聚焦显微镜显示，VAT-1修饰了细胞外围的颗粒结构，VAT-1/乳铁蛋白抗体的双重标记显示，在对照组和FMLF刺激的细胞中，VAT-1与次级颗粒部分共存。这些推测的PA结合蛋白的特征构成了更好地理解PLD来源的PA在中性粒细胞生理学中的作用的又一步。(C)2010 Elsevier Inc.保留所有权利。
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. (C) 2010 Elsevier Inc. All rights reserved.