Debreceni Balazs, Gao Yuan, Guo Fukun, Zhu Kejin, Jia Baoqing, Zheng Yi
Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
J Biol Chem. 2004 Jan 30;279(5):3777-86. doi: 10.1074/jbc.M308282200. Epub 2003 Nov 3.
Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis. Dominant negative mutants of Rho GTPases, such as T17NRac1, that block the endogenous Rho protein activation by sequestering upstream guanine nucleotide exchange factors (GEFs) have been widely used to implicate specific members of the Rho family in various signaling pathways. We show here that such an approach could produce potentially misleading results since many Rho GEFs can interact with multiple Rho proteins promiscuously, and overexpression of one dominant negative Rho protein mutant may affect the activity of other members of the Rho family. Based on the available structural information, we have identified the highly conserved amino acid pairing of Asn(1406)Trio-Asp(65)Rac1 of the GEF-Rho GTPase interaction as the critical catalytic machinery required for the Rac1 GDP/GTP exchange reaction. The N1406A/D1407A mutant of Trio acted dominant negatively in vitro by retaining Rac1 binding activity but losing GEF catalytic activity and competitively inhibited Rac1 activation by wild type Trio. It readily blocked the platelet-derived growth factor (PDGF)-induced lamellipodia formation and inhibited the wild type Trio-induced serum response factor activation. Moreover the mutant was able to selectively inhibit Dbl-induced Rac1 activation without affecting RhoA activity in cells. In contrast to the non-discriminative inhibitory effect displayed by T17NRac1, the Trio mutant was ineffective in inhibiting PDGF-stimulated DNA synthesis and Dbl-induced transformation, revealing the Rac-independent functions of PDGF and Dbl. These studies identify a conserved pair of amino acid residues of the Trio-Rac interaction that is likely to be essential to the GEF catalysis of Rho family GTPases and demonstrate that a dominant negative mutant derived from a Rho GTPase regulator constitutes a new generation of specific inhibitors of Rho GTPase signaling pathways.
Rho家族GTP酶在多种细胞过程中发挥重要作用，包括肌动蛋白细胞骨架重组、转录激活和DNA合成。Rho GTP酶的显性负性突变体，如T17NRac1，通过隔离上游鸟嘌呤核苷酸交换因子（GEF）来阻断内源性Rho蛋白的激活，已被广泛用于阐明Rho家族的特定成员在各种信号通路中的作用。我们在此表明，这种方法可能会产生潜在的误导性结果，因为许多Rho GEF可以与多种Rho蛋白随意相互作用，并且一种显性负性Rho蛋白突变体的过表达可能会影响Rho家族其他成员的活性。基于现有的结构信息，我们确定了GEF-Rho GTP酶相互作用中Asn(1406)Trio-Asp(65)Rac1的高度保守氨基酸配对，这是Rac1 GDP/GTP交换反应所需的关键催化机制。Trio的N1406A/D1407A突变体在体外表现出显性负性作用，它保留了Rac1结合活性但失去了GEF催化活性，并竞争性抑制野生型Trio对Rac1的激活。它很容易阻断血小板衍生生长因子（PDGF）诱导的片状伪足形成，并抑制野生型Trio诱导的血清反应因子激活。此外，该突变体能够在细胞中选择性抑制Dbl诱导的Rac1激活，而不影响RhoA活性。与T17NRac1表现出的无差别抑制作用相反，Trio突变体在抑制PDGF刺激的DNA合成和Dbl诱导的转化方面无效，这揭示了PDGF和Dbl的Rac非依赖性功能。这些研究确定了Trio-Rac相互作用中一对保守的氨基酸残基，它们可能对Rho家族GTP酶的GEF催化至关重要，并证明源自Rho GTP酶调节剂的显性负性突变体构成了新一代Rho GTP酶信号通路的特异性抑制剂。
