Malcolm K C, Ross A H, Qiu R G, Symons M, Exton J H
Howard Hughes Medical Institute, Nashville, Tennessee.
J Biol Chem. 1994 Oct 21;269(42):25951-4.
Stimulation of phospholipase D by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in rat liver plasma membranes indicates the involvement of GTP-binding proteins. We used RhoGDI, an inhibitor of GDP dissociation from small GTP-binding proteins of the Rho family, to determine the involvement of these proteins. Incubation, and subsequent washing, of plasma membranes with RhoGDI dose-dependently diminished GTP gamma S-stimulated phospholipase D activity, as determined by accumulation of phosphatidylethanol in the presence of ethanol. Incubation with RhoGDI also caused a rapid and dose-dependent appearance of RhoA in the wash, which was associated with the inhibition of phospholipase D. RhoGDI also rapidly extracted Cdc42 from membranes, but Rac1 was not extracted. Full reconstitution of GTP gamma S-stimulated phospholipase D in RhoGDI-washed membranes was achieved with recombinant RhoA. There was partial reconstitution with Rac1 and no enhancement with Cdc42 or ADP-ribosylation factor. The response to RhoA was dose-dependent (EC50 = 0.5 microM). ADP-ribosylation of RhoA by Clostridium botulinum C3 exoenzyme did not affect its ability to recover GTP gamma S-stimulated phospholipase D activity in RhoGDI-washed membranes. These findings support a role for GTP-binding proteins of the Rho family in the activation of membrane-associated phospholipase D and implicate RhoA as the major protein involved.
鸟苷5'-O-(3-硫代三磷酸)(GTPγS)对大鼠肝细胞膜中磷脂酶D的刺激表明GTP结合蛋白参与其中。我们使用RhoGDI（一种抑制GDP从小GTP结合蛋白Rho家族解离的抑制剂）来确定这些蛋白的参与情况。用RhoGDI孵育并随后洗涤细胞膜，GTPγS刺激的磷脂酶D活性呈剂量依赖性降低，这是通过在乙醇存在下磷脂酰乙醇的积累来确定的。用RhoGDI孵育还导致洗涤液中RhoA迅速且呈剂量依赖性出现，这与磷脂酶D的抑制相关。RhoGDI也能迅速从膜中提取Cdc42，但不能提取Rac1。用重组RhoA可使RhoGDI洗涤过的膜中GTPγS刺激的磷脂酶D完全恢复。Rac1可部分恢复，而Cdc42或ADP核糖基化因子则无增强作用。对RhoA的反应呈剂量依赖性(EC50 = 0.5 microM)。肉毒杆菌C3外毒素对RhoA的ADP核糖基化不影响其在RhoGDI洗涤过的膜中恢复GTPγS刺激的磷脂酶D活性的能力。这些发现支持Rho家族的GTP结合蛋白在膜相关磷脂酶D激活中的作用，并表明RhoA是主要参与的蛋白。
