Malcolm K C, Elliott C M, Exton J H
Howard Hughes Medical Institute and the Departments of Molecular Physiology and Biophysics and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
J Biol Chem. 1996 May 31;271(22):13135-9. doi: 10.1074/jbc.271.22.13135.
Small GTP-binding proteins of the Rho family are implicated in the in vitro regulation of phosphatidylcholine hydrolysis by phospholipase D (PLD). However, their role in agonist-stimulated PLD activity in whole cells is not clear. The ribosyltransferase C3 from Clostridium botulinum modifies Rho proteins and inhibits their function. When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity stimulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol ester. Neither the time course nor agonist dose response for LPA-stimulated PLD activity was altered in C3-treated cells. In contrast to the effects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-phospholipase C activity was not altered in C3-treated cells. Surprisingly, C3 treatment led to a decrease in the amount of RhoA protein, indicating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins. As described previously, C3 treatment led to the inhibition of LPA-stimulated actin filament formation. However, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity. Interestingly, stimulation of cells with LPA caused a rapid enrichment of RhoA in the particulate fraction of cell lysates. These data support an in vivo role for RhoA in agonist-stimulated PLD activity that is separate from its role in actin fiber formation.
Rho家族的小GTP结合蛋白与磷脂酶D（PLD）在体外对磷脂酰胆碱水解的调节有关。然而，它们在全细胞中激动剂刺激的PLD活性中的作用尚不清楚。肉毒杆菌的核糖基转移酶C3修饰Rho蛋白并抑制其功能。通过刮涂加载法将C3导入大鼠1成纤维细胞后，C3抑制了溶血磷脂酸（LPA）、内皮素-1或佛波酯刺激的PLD活性。在C3处理的细胞中，LPA刺激的PLD活性的时间进程和激动剂剂量反应均未改变。与C3对PLD活性的影响相反，在C3处理的细胞中，激动剂刺激的磷脂酰肌醇-磷脂酶C活性未改变。令人惊讶的是，C3处理导致RhoA蛋白量减少，表明对激动剂反应时PLD活性的丧失部分归因于Rho蛋白的丧失。如先前所述，C3处理导致LPA刺激的肌动蛋白丝形成受到抑制。然而，用细胞松弛素D破坏肌动蛋白丝仅对LPA刺激的PLD活性产生轻微抑制。有趣的是，用LPA刺激细胞会导致RhoA在细胞裂解物的颗粒部分迅速富集。这些数据支持RhoA在激动剂刺激的PLD活性中具有体内作用，这与其在肌动蛋白纤维形成中的作用是分开的。
