The Verprolin-like Central (VC) region of Wiskott-Aldrich syndrome protein induces Arp2/3 complex-dependent actin nucleation
The Verprolin-like Central (VC) region of Wiskott-Aldrich syndrome protein induces Arp2/3 complex-dependent actin nucleation
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DOI：
10.1074/jbc.m106520200
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发表时间：
2001-09-21
影响因子：
4.8
通讯作者：
Linder, S
中科院分区：
文献类型：
--
作者：
Hüfner, K;Higgs, HN;Linder, S
Wiskott-Aldrich综合征蛋白（WASp）及其相关蛋白通过Arp 2/3复合物刺激肌动蛋白丝成核。WASp的分离的C-末端VCA结构域（含有Yerprolin样、中央和酸性区域）是组成型活性的，但在全长蛋白中是自抑制的。本研究比较了部分VCA融合到谷胱甘肽S-转移酶（GST）的C末端，在体外结合肌动蛋白和Arp 2/3复合物，并在体外和细胞中激活肌动蛋白聚合的能力。荧光各向异性测量显示GST-CA和GST-A结合Arp 2/3复合物的Kd值分别为0.11 μ M和1.0 μ M，而GST-VC显示几乎检测不到结合（Kd> 1 mm）。然而，GST-VC在体外通过Arp 2/3复合物激活肌动蛋白成核，尽管需要比GST-VCA高70倍的浓度，而GST-CA和GST-A在体外都不激活Arp 2/3复合物，尽管GST-CA和GST-A都抑制WASp VCA激活Arp 2/3复合物。这些构建体均不结合来自巨噬细胞裂解物的WASp。GST-VC和GST-CA都诱导肌动蛋白的积累时，显微注射到原代人巨噬细胞或人内皮静脉细胞。然而，只有微量注射GST-VC导致细胞聚合肌动蛋白的显着增加。此外，内源性Arp 2/3复合物，但不是WASp，共定位与这些GST-VC诱导的肌动蛋白积累。这些数据表明，WASp构建体缺乏A区，以前认为是必不可少的肌动蛋白成核，能够结合和激活Arp 2/3复合物在体外和体内。
Wiskott-Aldrich Syndrome protein (WASp) and related proteins stimulate actin filament nucleation by Arp2/3 complex. The isolated C-terminal VCA domain of WASp (containing Yerprolin-like, Central and Acidic regions) is constitutively active but autoinhibited in the full-length protein. This study compared the ability of parts of VCA fused to the C terminus of glutathione S-transferase (GST) to bind actin and Arp2/3 complex in vitro and to activate actin polymerization in vitro and in cells. Fluorescence anisotropy measurements showed that GST-CA and GST-A bound Arp2/3 complex with K-d values of 0.11 muM and 1.0 muM, respectively, whereas GST-VC displayed almost undetectable binding (K-d > I mm). However, GST-VC activated actin nucleation through Arp2/3 complex in vitro, though requiring 70-fold higher concentration than GST-VCA while neither GST-CA nor GST-A activated Arp2/3 complex in vitro, though both GST-CA and GST-A inhibited Arp2/3 complex activation by WASp VCA. None of these constructs bound WASp from macrophage lysates. Both GST-VC and GST-CA induced actin accumulations when microinjected into primary human macrophages or human endothelial vein cells. However, only microinjection of GST-VC led to a significant increase of cellular polymerized actin. Additionally, endogenous Arp2/3 complex, but not WASp, colocalized with these GST-VC-induced actin accumulations. These data suggest that WASp constructs lacking the A region, previously thought to be indispensable for actin nucleation, are able to bind and activate Arp2/3 complex in vitro and in vivo.