Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments
Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments
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DOI：
10.1073/pnas.261419298
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发表时间：
2001-12-18
影响因子：
11.1
通讯作者：
Mullins, RD
中科院分区：
文献类型：
--
作者：
Dayel, MJ;Holleran, EA;Mullins, RD
Arp2/3复合物是一种含有两个肌动蛋白相关蛋白Arp2和Arp3的七亚基蛋白复合物，它响应细胞内信号启动肌动蛋白丝网络的形成。然而，细丝成核的分子机制还没有很好地理解。Arp2和Arp3被预测通过在肌动蛋白超家族的所有成员中发现的高度保守的核苷酸结合结构域结合ATP，并形成异源二聚体而不是模拟常规的肌动蛋白二聚体。我们在这里表明，腺苷核苷酸以微摩尔亲和力与Arp 2和Arp 3结合，并且肌动蛋白成核活性需要可水解的ATP。N-WASP WA的结合增加了Arp2和Arp3对ATP的亲和力，但不改变在饱和浓度的ATP存在下结合的核苷酸的化学计量。Arp 2/3复合物与ADP或不可水解的ATP类似物AMP-PNP结合不能使肌动蛋白丝成核，但加入磷酸盐类似物BeF 3可部分恢复ADP-Arp 2/3复合物的活性。结合的核苷酸还调节Arp 2/3复合物对其上游活化剂N-WASP和ActA的亲和力。我们建议，活性成核形式的Arp 2/3复合物是ADP-Pi的ATP酶循环中的中间体，并且Arp 2/3复合物的ATP酶活性控制新丝的成核和Arp 2/3复合物从膜相关激活剂的释放。
The Arp2/3 complex, a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, initiates formation of actin filament networks in response to intracellular signals. The molecular mechanism of filament nucleation, however, is not well understood. Arp2 and Arp3 are predicted to bind ATP via a highly conserved nucleotide-binding domain found in all members of the actin superfamily and to form a heterodimer than mimics a conventional actin dimer. We show here that adenosine nucleotides bind with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable ATP is required for actin nucleation activity. Binding of N-WASP WA increases the affinity of both Arp2 and Arp3 for ATP but does not alter the stoichiometry of nucleotides bound in the presence of saturating concentrations of ATP. The Arp2/3 complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments, but addition of the phosphate analogue BeF3 partially restores activity to the ADP-Arp2/3 complex. Bound nucleotide also regulates the affinity of the Arp2/3 complex for its upstream activators N-WASP and ActA. We propose that the active nucleating form of the Arp2/3 complex is the ADP-Pi intermediate in the ATPase cycle and that the ATPase activity of the Arp2/3 complex controls both nucleation of new filaments and release of the Arp2/3 complex from membrane-associated activators.