LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEINS IN HL-60 GRANULOCYTES - ASSESSMENT OF THE ROLE OF ARF AND OF A 50-KDA CYTOSOLIC PROTEIN IN PHOSPHOLIPASE-D ACTIVATION
LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEINS IN HL-60 GRANULOCYTES - ASSESSMENT OF THE ROLE OF ARF AND OF A 50-KDA CYTOSOLIC PROTEIN IN PHOSPHOLIPASE-D ACTIVATION
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DOI：
10.1074/jbc.270.7.3172
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发表时间：
1995-02-17
影响因子：
4.8
通讯作者：
BEAULIEU, A
中科院分区：
文献类型：
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作者：
BOURGOIN, S;HARBOUR, D;BEAULIEU, A
鸟嘌呤核苷酸激活磷脂酶D（PLD）需要质膜和胞浆中的蛋白质辅因子。HL-60胞浆用硫酸铵和凝胶渗透色谱法分级，发现两种胞浆蛋白组分可重建GTP γ S（鸟苷5 ′-3-O-（硫代）三磷酸）刺激的PLD在由H-3-标记的HL-60膜和洗脱的柱级分组成的重建测定中的变化。重组活性的主峰在50 kDa区域，PLD重组活性的第二个离散峰在18 kDa区域，Rho GDP/GTP交换抑制剂Rho GDI与Rac 2和RhoA共迁移，但不与Rad共迁移。RhoA和Rac 2与Rho GDI完全复合，并通过凝胶过滤色谱以43 kDa的表观分子量洗脱。胞质Rac 2和RhoA之间的部分重叠与50 kDa的重组活性峰不一致，这与胞质Rho相关的GTP酶参与鸟嘌呤核苷酸激活PLD不一致。然而，重组Rho GDI，它抑制小GTP结合蛋白的Rho家族的核苷酸交换，减少GTP γ S刺激的PLD活性在HL-60匀浆。通过凝胶渗透色谱法可以将对Rho和Rac具有活性的刺激性交换因子Smg GDS与PLD刺激因子部分分离。重组SmgGDS不能刺激GTP依赖的PLD活性，胞浆ADP核糖基化因子（ARF）仅位于18 kDa的重构活性峰，单克隆抗体1D 9也检测到少量的膜结合ARF。50 kDa和18 kDa PLD诱导因子对盐提PLD活性的影响具有协同作用。ARF单独的弱刺激作用表明GTP γ S刺激的PLD活性依赖于另一种蛋白质的存在，可能是ARF调节蛋白。我们提出，一个膜结合的GTP结合蛋白，可能ARF，可能参与PLD的激活时，结合的组件（S）的50 kDa的馏分。
Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol, HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromotography, Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) stimulated PLD in a reconstitution assay consisting of H-3-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa, Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rad. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography, The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP dependent PLD activity, Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity, Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9, The effects of the 50-kDa and 18-kDa PLD inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50 kDa fraction.