Marcil J, Harbour D, Naccache P H, Bourgoin S
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Ste-Foy, Québec G1V 4G2, Canada.
J Biol Chem. 1997 Aug 15;272(33):20660-4. doi: 10.1074/jbc.272.33.20660.
The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.
人磷脂酶D1（hPLD1）最近已被克隆。尽管最近的数据表明PLD参与受体刺激的分泌，但PLD酶活性的调节仍有待阐明。纯化的hPLD1可被几种胞质辅因子激活，其中包括蛋白激酶Cα、ARF和RhoA。在人粒细胞中，已建立蛋白质酪氨酸磷酸化与PLD活性之间的强相关性。在本研究中，对HL-60粒细胞中hPLD1的存在及其酪氨酸残基上的磷酸化进行了研究。我们制备了对hPLD1特异而对PLD2不特异的抗肽抗体（Abs），重组PLD1和PLD2的蛋白质印迹（WB）结果表明了这一点。这些抗体鉴定出HL-60细胞中存在hPLD1，其中大部分在膜中检测到，只有一小部分在胞质溶胶中。hPLD1抗体在120 kDa处检测到一条主要条带（PLD1a），在115 kDa处检测到一条次要条带（PLD1b）。用免疫原性肽中和的PLD抗血清证实了抗体的特异性。在用hPLD1抗体进行WB分析后，在非变性和变性条件下通过免疫沉淀始终检测到两种形式的hPLD1。在将HL-60细胞暴露于酪氨酸磷酸酶抑制剂钒酸盐过氧化物（V4+-OOH）后，在非变性条件下从HL-60细胞裂解物中免疫沉淀hPLD1，并通过WB检测酪氨酸磷酸化。通过凝胶电泳，hPLD1与一种120 kDa的酪氨酸磷酸化蛋白共迁移。在hPLD1免疫复合物中还检测到其他160、140、135、90和75 - 80 kDa的酪氨酸磷酸化肽段。hPLD1和相关的酪氨酸磷酸化蛋白未被中和的hPLD1抗体免疫沉淀。在变性条件下，将PLD免疫沉淀物依次用抗PLD1和抗磷酸酪氨酸抗体进行免疫印迹。检测到PLD1a和PLD1b，主要的PLD1a蛋白与在120 kDa处检测到的主要酪氨酸磷酸化蛋白重叠。相反，PLD1a和PLD1b至少部分在抗磷酸酪氨酸免疫沉淀物中回收。这些结果提供了证据，表明在人粒细胞中表达两种PLD1形式。此外，响应V4+-OOH的刺激，PLD1发生酪氨酸磷酸化，并与几种目前未定义的酪氨酸磷酸化蛋白相关。
