Abstract
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.
Highlights
Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis
Given the recent realization that many Dbl family guanine nucleotide exchange factors (GEFs), the core Dbl homology (DH)-Pleckstrin homology (PH) module that is responsible for Rho GTPase recognition and catalysis, are promiscuous in nature [5, 6], it could be expected that overexpression of one dominant negative Rho GTPase might indiscriminately inhibit other Rho GTPase activities by sequestering multiple GEFs or one GEF that is capable of activating multiple Rho protein substrates, leading to incorrect conclusions
Administration of Dominant Negative Rac1 Could Nonspecifically Affect RhoA Activity in Cells—The dominant negative mutants of Rac1 and Cdc42, T17NRac1 and T17NCdc42, are widely used to inhibit Rac- or Cdc42-regulated signaling pathways. They were derived based on the corresponding mutant of Ras, which is capable of sequestering Ras-specific GEFs and blocks endogenous Ras activity [15]. These mutants were shown to bind to the Dbl-like GEFs with high affinity in vitro [16] and to inhibit Rac-mediated lamellipodia formation and membrane ruffling or Cdc42-mediated filopodia induction in vivo [10, 26, 27] Given the apparent existence of a large number of Dbl family GEFs in mammalian cells and the realization that many of them can bind to and activate multiple Rho proteins [5], we were concerned that non-discriminative application of these dominant negative Rho mutants in cells where abundant GEFs are expressed may produce misleading results
Summary
Ruffling elicited by PDGF [10], whereas dominant negative RhoA, Rac, and Cdc could all inhibit serum response factor activation and G1/S phase transition of the cell cycle [11] and Ras-induced cell transformation [12,13,14]. The proposed mechanism of the dominant negative mutant function is that it binds to its respective GEFs with high affinity and may sequester the endogenous Rho GEFs by forming a non-functional dominant negative Rho-GEF complex [15, 16]. It is often assumed by users of these mutants that a given mutant blocks only the specific pathway(s) that is activated by its wild type counterpart. Our results indicate that dominant negative mutants derived from a Rho GTPase regulator may constitute a new generation of specific inhibitors of Rho GTPase signaling pathways
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