Direct binding of SWAP-70 to non-muscle actin is required for membrane ruffling
Direct binding of SWAP-70 to non-muscle actin is required for membrane ruffling
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DOI：
10.1242/jcs.02767
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发表时间：
2006-02-01
影响因子：
4
通讯作者：
Fukui, Y
中科院分区：
文献类型：
--
作者：
Ihara, S;Oka, T;Fukui, Y
生长因子刺激引起的膜褶皱是由肌动蛋白重塑引起的，肌动蛋白重塑是由包括 Rac 在内的多种信号分子介导的。我们已经证明，与磷脂酰肌醇三磷酸结合的 SWAP-70 是小鼠肾细胞膜褶皱所需的分子之一。在这里，我们证明 SWAP-70 直接与 F-肌动蛋白结合。细菌表达的 SWAP-70 C 末端区域与非肌肉 F-肌动蛋白共沉淀，表明 SWAP-70 与 F-肌动蛋白直接结合。肌肉 F-肌动蛋白中的结合要弱得多。仅包含 C 末端区域的 SWAP-70 截短突变体很容易与 F-肌动蛋白共定位，支持了这一想法。除非用生长因子刺激细胞，否则全长 SWAP-70 不会与 F-肌动蛋白共定位，这表明体内肌动蛋白结合活性存在刺激依赖性调节机制。缺乏该结合域的突变体 SWAP-70 的过表达会抑制 COS7 细胞中表皮生长因子刺激引起的膜皱褶。这种显性负效应也在显性活性 Rac 诱导的膜波纹中观察到，表明 SWAP-70 与 Rac 合作。这些结果表明 SWAP-70 与非肌肉 F-肌动蛋白的结合活性是膜褶皱所必需的。
Membrane ruffling induced by growth factor stimulation is caused by actin remodeling, which is mediated by various signaling molecules including Rac. We have shown that SWAP-70, which binds phosphatidylinositol trisphosphate, is one such molecule required for membrane ruffling in mouse kidney cells. Here, we show that SWAP-70 directly binds to F-actin. The bacterially expressed C-terminal region of SWAP-70 co-sedimented with non-muscle F-actin, suggesting direct binding of SWAP-70 to F-actin. The binding was much weaker in muscle F-actin. A truncated mutant of SWAP-70 containing only the C-terminal region readily colocalizes with F-actin, supporting this idea. Full-length SWAP-70 does not colocalize with F-actin unless cells are stimulated with growth factors, suggesting the presence of a stimuli-dependent regulatory mechanism for actin-binding activity in vivo. Overexpression of the mutant SWAP-70 lacking this binding domain inhibits the membrane ruffling induced by epidermal growth factor stimulation in COS7 cells. This dominant-negative effect is also observed in membrane ruffling induced by a dominant-active Rac, suggesting that SWAP-70 cooperates with Rac. These results suggest that the binding activity of SWAP-70 to non-muscle F-actin is required for membrane ruffling.