Kim Kyoungtae, Hou Peng, Gorski Jerome L, Cooper John A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2004 Mar 9;43(9):2422-7. doi: 10.1021/bi036173t.
Mutations in faciogenital dysplasia protein (Fgd1) result in the human disease faciogenital dysplasia (FGDY). Fgd1 contains a RhoGEF domain specific for Cdc42. Fgd1 also contains a Src homology (SH3) binding domain (SH3-BD) that binds directly to the SH3 domain of cortactin, which promotes actin assembly by actin-related protein (Arp)2/3 complex. Here, we report the effect of ligation of cortactin's SH3 domain by the Fgd1 SH3-BD on actin polymerization in vitro. Glutathione S-transferase (GST)-fused Fgd1 SH3-BD enhanced the ability of cortactin to stimulate Arp2/3-mediated actin polymerization. However, a synthetic peptide containing only the SH3-BD sequence had no effect. The SH3-BD peptide bound to cortactin and inhibited the effect of GST-Fgd1 SH3-BD, suggesting that GST dimerization was responsible for the stimulating effect of GST-Fgd1 SH3-BD. When GST-Fgd1 SH3-BD was prepared as a heterodimer with a control GST fusion protein (GST-Pac1), no stimulatory effect on actin polymerization was observed. In addition, when cortactin was dimerized via its N-terminus, away from the C-terminal SH3 domain, actin polymerization with Arp2/3 complex increased markedly, compared to free cortactin. Thus, cortactin ligated by Fgd1 is fully active, indicating that the cell can use Fgd1 to target actin assembly. Moreover, if Fgd1 is multimerized, then cortactin's activity should be enhanced. Fgd1 and cortactin may participate as scaffolds and signal transducers in a positive feedback cycle to promote actin assembly at the cell cortex.
面部生殖器发育不全蛋白（Fgd1）的突变会导致人类疾病面部生殖器发育不全（FGDY）。Fgd1包含一个对Cdc42具有特异性的Rho鸟苷酸交换因子（RhoGEF）结构域。Fgd1还包含一个Src同源（SH3）结合结构域（SH3-BD），该结构域直接与cortactin的SH3结构域结合，cortactin通过肌动蛋白相关蛋白（Arp）2/3复合物促进肌动蛋白组装。在此，我们报告了Fgd1的SH3-BD对cortactin的SH3结构域的连接在体外对肌动蛋白聚合的影响。谷胱甘肽S-转移酶（GST）融合的Fgd1 SH3-BD增强了cortactin刺激Arp2/3介导的肌动蛋白聚合的能力。然而，仅包含SH3-BD序列的合成肽没有作用。SH3-BD肽与cortactin结合并抑制了GST-Fgd1 SH3-BD的作用，表明GST二聚化是GST-Fgd1 SH3-BD产生刺激作用的原因。当将GST-Fgd1 SH3-BD与对照GST融合蛋白（GST-Pac1）制备成异二聚体时，未观察到对肌动蛋白聚合的刺激作用。此外，当cortactin通过其N端二聚化，远离C端的SH3结构域时，与游离的cortactin相比，与Arp2/3复合物的肌动蛋白聚合显著增加。因此，被Fgd1连接的cortactin具有完全活性，表明细胞可以利用Fgd1靶向肌动蛋白组装。此外，如果Fgd1多聚化，那么cortactin的活性应该会增强。Fgd1和cortactin可能作为支架和信号转导分子参与一个正反馈循环，以促进细胞皮层的肌动蛋白组装。
