ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils
ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils
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DOI：
10.1074/jbc.273.21.13157
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发表时间：
1998-05-22
影响因子：
4.8
通讯作者：
Cockcroft, S
中科院分区：
文献类型：
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作者：
Fensome, A;Whatmore, J;Cockcroft, S
fMLP激活完整的人中性粒细胞，通过未知的信号通路刺激磷脂酶D （PLD）。小GTPase、adp -核糖基化因子（ARF）和Rho蛋白直接调节PLD1的活性。溶血素O对细胞的渗透性会导致包括ARF在内的胞质蛋白的丢失，但不会导致中性粒细胞中Rho蛋白的丢失，fMLP在这些胞质枯竭的细胞中难以激活PLD。激活myr-ARF1而非myr-ARF1可以恢复fMLP刺激的PLD活性，C3毒素使Rho蛋白失活，降低了ARF重建的PLD活性，说明虽然Rho单独不刺激PLD活性，但它与ARF协同作用。为了确定fMLP激活ARF和Rho的信号通路，我们使用百日毒和wortmannin分别检测了对G(i)家族异三聚体G蛋白和磷酸肌肽3-激酶的需求。百日咳毒素抑制了完整细胞中的PLD活性，也抑制了细胞质枯竭细胞中arf恢复反应的PLD活性，表明需要G(i2)/G（i3）蛋白。相反，wortmannin仅抑制fmlp刺激的完整中性粒细胞PLD活性，但对myr- arf1重组活性没有影响。由此可见，fMLP激活ARF/Rho必须激活G(i)蛋白，而不需要激活磷酸肌苷3-激酶。
Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads Do loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils, PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity, C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF, To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the G(i) family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for G(i2)/G(i3) protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin, It is concluded that activation of G(i) proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.