Identification of Rho GTPase-dependent Sites in the Dbl Homology Domain of Oncogenic Dbl That Are Required for Transformation

Abstract

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the ␣1, ␣6, and ␣9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k cat values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42-and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.

Mechanisms of Guanine Nucleotide Exchange and Rac-mediated Signaling Revealed by a Dominant Negative Trio Mutant

“…cDNA Constructs-The DH domain point mutants of TrioN (T1244A, N1406A, and N1406A/D1407A; residues 1225-1537 of Trio) were generated by oligonucleotide-directed mutagenesis of human Trio cDNA in pET15b vector by the polymerase chain reaction-based second extension amplification technique using the Pfu polymerase (Stratagene) with primers that contained the desired mutations (17). The sequences of mutagenized cDNA inserts were confirmed by automated DNA sequencing.…”

“…The sequences of mutagenized cDNA inserts were confirmed by automated DNA sequencing. The BamHI-EcoRI fragments encoding the DH-PH module of wild type or mutant TrioN were subsequently subcloned into the corresponding sites of pMX-IRES-GFP retroviral vector for recombinant retroviral production or pCEFL-GST vector for transient transfection (17,18).…”

“…Mutants in Cells-Wild type, N1406A/D1407A, and T1244A of TrioN, T17NRac1, and T17NCdc42 were expressed in NIH 3T3 or Swiss 3T3 cells by using the pMX-IRES-GFP vector, which expresses the green fluorescent protein (GFP) as a bicistronic mRNA (17,21). Retroviral packaging and infection were carried out according to the described methods (22).…”

“…In Vivo Rac1 and RhoA GTPase Activation Assay-The onco-Dbl-expressing cells were generated previously (17,18). The glutathione-agarose-immobilized GST-PAK1, which contains the p21-binding domain of human PAK1 (residues 51-149), and the GST-Rhotekin, which contains the site required for RhoA-GTP recognition of Rhotekin (residues 7-89), were expressed and purified in E. coli by using the pGEX-KG vector described previously (23,24).…”

“…Coverslips were mounted onto slides in 50% glycerol, Tris-buffered saline. Stained cells were analyzed by using a Leica confocal fluorescence microscope (17).…”