Cheng Li, Mahon Gwendolyn M, Kostenko Elena V, Whitehead Ian P
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 225 Warren Street, Newark, NJ 07103, USA.
J Biol Chem. 2004 Mar 26;279(13):12786-93. doi: 10.1074/jbc.M313099200. Epub 2003 Dec 30.
Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose expression causes deregulated growth in NIH 3T3 mouse fibroblasts. Although Rac1 has not been shown to be a substrate for Dbs in either in vitro or in vivo assays, the Rat ortholog of Dbs (Ost) has been shown to bind specifically to GTP.Rac1 in vitro. The dependence of the Rac1/Dbs interaction on GTP suggests that Dbs may in fact be an effector for Rac1. Here we show that the interaction between activated Rac1 and Dbs can be recapitulated in mammalian cells and that the Rac1 docking site resides within the pleckstrin homology domain of Dbs. This interaction is specific for Rac1 and is not observed between Rac1 and several other members of the Rho-specific guanine nucleotide exchange factor family. Co-expression of Dbs with activated Rac1 causes enhanced focus forming activity and elevated levels of GTP.RhoA in NIH 3T3 cells, indicating that Dbs is activated by the interaction. Consistent with this, activated Rac1 co-localizes with Dbs in NIH 3T3 cells, and natively expressed Rac1 relocalizes in response to Dbs expression. To summarize, we have characterized a surprisingly direct pleckstrin homology domain-mediated mechanism through which Rho GTPases can become functionally linked.
Dbs是一种Rho特异性鸟嘌呤核苷酸交换因子，它是在对那些表达会导致NIH 3T3小鼠成纤维细胞生长失调的蛋白质进行筛选时被鉴定出来的。尽管在体外或体内实验中均未表明Rac1是Dbs的底物，但Dbs的大鼠直系同源物（Ost）已被证明在体外能特异性结合GTP·Rac1。Rac1与Dbs的相互作用对GTP的依赖性表明，Dbs实际上可能是Rac1的效应器。在此我们表明，活化的Rac1与Dbs之间的相互作用可在哺乳动物细胞中重现，且Rac1的对接位点位于Dbs的普列克底物蛋白同源结构域内。这种相互作用对Rac1具有特异性，在Rac1与Rho特异性鸟嘌呤核苷酸交换因子家族的其他几个成员之间未观察到。Dbs与活化的Rac1共表达会导致NIH 3T3细胞中集落形成活性增强以及GTP·RhoA水平升高，这表明Dbs通过这种相互作用被激活。与此一致的是，活化的Rac1在NIH 3T3细胞中与Dbs共定位，并且天然表达的Rac1会响应Dbs的表达而重新定位。总之，我们已经描述了一种惊人的直接的普列克底物蛋白同源结构域介导的机制，通过该机制Rho GTP酶可以在功能上建立联系。
