Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle
Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle
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DOI：
10.1152/ajpcell.00387.2004
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发表时间：
2005-05-01
影响因子：
5.5
通讯作者：
Gunst, SJ
中科院分区：
文献类型：
--
作者：
Zhang, WW;Wu, YD;Gunst, SJ
收缩刺激已经显示在平滑肌组织中启动肌动蛋白聚合，并且这种肌动蛋白聚合是主动张力发展所需的。我们评估了神经元Wiskott-Aldrich综合征蛋白（N-WASp）介导的肌动蛋白相关蛋白2和3（Arp 2/3）复合物激活是否调节犬气管平滑肌组织中由毒蕈碱刺激引发的肌动蛋白聚合和张力发展。在体外，N-WASp的COOH-末端CA结构域作为N-WASp介导的肌动蛋白聚合的抑制剂;而N-WASp的COOH-末端VCA结构域是组成型活性的，并且本身足以催化肌动蛋白聚合。通过可逆透化将编码EGFP标记的野生型N-WASp、N-WASp VCA和CA结构域或增强的绿色荧光蛋白（EGFP）的质粒引入气管平滑肌条，并将组织孵育2天以允许蛋白质的表达。CA域的表达抑制肌动蛋白聚合和张力发展响应ACh，而野生型N-WASp，VCA域，或EGFP的表达没有。肌球蛋白轻链（MLC）磷酸化反应的收缩刺激的增加不受CA或VCA结构域的N-WASp的表达。用ACh刺激组织增加Arp 2/3复合物与N-WASp的关联，并且这种关联被CA结构域的表达抑制。结果表明，1）N-WASp介导的Arp 2/3复合物的活化是响应于气管平滑肌中的毒蕈碱刺激的肌动蛋白聚合和张力发展所必需的，并且2）这些作用不依赖于MLC磷酸化的调节。
Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation.