Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
Biochemistry. 2011 Feb 15;50(6):1070-80. doi: 10.1021/bi100971y. Epub 2011 Jan 20.
Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.
鸟嘌呤核苷酸交换因子（GEFs）通过催化结合的 GDP 与 GTP 的交换来激活 Rho GTPases，从而导致下游效应物的识别。已经描述了两种后生动物家族的 GEFs：共享保守的 Dbl 同源性（DH）和 Pleckstrin 同源性（PH）结构域的 Dbl-GEF 家族成员，以及最近描述的共享与 Dbl 家族很少序列同源性的 Dock180 家族成员，其特征是保守的 Dock 同源结构域 1 和 2（分别为 DHR-1 和 -2）。虽然已经对 Dbl 家族进行了广泛的表征，但对于 Dock180 家族成员如何作为 GEFs 发挥作用的了解较少，只有最近才报道了一个 Dock9-Cdc42 复合物的 X 射线结构。为了更多地了解家族的创始成员 Dock180 作为 Rac 特异性 GEF 的作用机制，我们着手确定并表征其限制功能的 GEF 结构域。DHR-2 结构域的 C 端部分，由大约 300 个残基组成（指定为 Dock180(DHR-2c)），在体外和体内均显示出对强大的 Rac 特异性 GEF 活性是必需和充分的。我们进一步表明，Dock180(DHR-2c)以与 Dbl 家族 Rac-GEFs 不同的方式结合 Rac。具体而言，Rac 的 Ala(27)和 Trp(56)似乎为 Dock180(DHR-2c)的特异性识别提供了一个二部分结合位点，而对于 Dbl 家族 Rac-GEFs，Rac 的 Trp(56)是 GEF 特异性的唯一主要决定因素。基于我们的发现，我们能够定义负责 Rac-GEF 活性的 Dock180 的核心，并突出区分不同 Dock180 家族成员并确定其相应 GTPase 特异性的关键识别位点。
