Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts, USA.
Cytoskeleton (Hoboken). 2010 Feb;67(2):120-33. doi: 10.1002/cm.20429.
Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K(d) approximately 1.5 microM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.
细胞过程由肌动蛋白聚合推动，需要快速解聚纤维，然后有效地将 ADF/cofilin 结合的 ADP-肌动蛋白单体循环回具有组装能力的 ATP 结合状态。单体再填充如何在体内受到调节尚不清楚，但最近的工作表明普遍存在的肌动蛋白单体结合蛋白 Srv2/CAP 的参与。为了更好地理解 Srv2/CAP 机制，我们探索了其 WH2 结构域的贡献，其功能仍然高度难以捉摸。我们发现 WH2 结构域结合肌动蛋白单体，并且与大多数其他 WH2 结构域不同，对 ATP-肌动蛋白和 ADP-肌动蛋白具有相似的结合亲和力（Kd 约为 1.5 μM）。破坏肌动蛋白结合的 WH2 结构域突变会破坏纯化全长 Srv2/CAP 催化 ADF/cofilin 结合的肌动蛋白单体上核苷酸交换的能力，并在体外加速肌动蛋白周转率。相同的突变会损害 Srv2/CAP 在体内调节肌动蛋白组织、细胞生长和细胞形态发生中的功能。因此，正常的细胞生长和组织依赖于 Srv2/CAP 为肌动蛋白单体再充电的能力，而 WH2 结构域在这个过程中起着核心作用。我们的数据还表明，虽然大多数分离的 WH2 结构域抑制肌动蛋白上的核苷酸交换，但完整蛋白中的 WH2 结构域可以帮助促进核苷酸交换。
