Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin
Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin
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DOI：
10.1074/jbc.274.39.27385
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发表时间：
1999-09-24
影响因子：
4.8
通讯作者：
Nozawa, Y
中科院分区：
文献类型：
--
作者：
Banno, Y;Fujita, H;Nozawa, Y
Gelsolin是一种肌动蛋白结合蛋白，具有很强的与磷脂酰肌醇4,5-二磷酸（PIP2）结合的能力。在体外实验中，我们发现gelsolin抑制重组磷脂酶D1 （PLD1）和PLD2的活性，但不抑制油酸依赖性PLD的活性，并且这种抑制不会通过增加PIP2浓度而逆转。为了研究凝胶蛋白在激动剂介导的PLD激活中的作用，我们使用NIH 3T3成纤维细胞稳定转染了人细胞质凝胶蛋白cDNA。Gelsolin过表达抑制缓激肽诱导的磷脂酶C （PLC）和PLD的激活。另一方面，鞘氨醇1-磷酸（S1P）诱导的PLD激活不能被凝胶蛋白过表达修饰，而PLC的激活被抑制。凝胶蛋白过表达不影响肉豆蔻酸酯或Ca2+离子载体A23187对PLD的激活。用缓激肽或S1P刺激对照细胞可引起蛋白激酶C （PKC）向细胞膜的易位。在凝胶蛋白过表达的细胞中，pkc - α和pkc - β 1的易位减少，但PKC-epsilon的易位没有减少，而丝裂原活化蛋白激酶的磷酸化没有改变。S1P诱导的PLC活化和丝裂原活化的蛋白激酶磷酸化对百日咳毒素敏感，但PLD反应对百日咳毒素不敏感，提示S1P诱导PLD活化的途径与PLC和丝裂原活化的蛋白激酶途径G(i)不同。我们的研究结果表明，凝胶胶通过抑制PLC和PKC活性来调节缓激肽介导的PLD激活，但不影响s1p介导的PLD激活。
Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP2). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP2 concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca2+ ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta 1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from G(i) for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.