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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 alpha1, alpha6, and alpha9 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.
Highlights
Many of the Dbl family members, such as Bcr, Cdc24, Sos, RasGRF, and Vav, have been known for their roles in cell growth regulations
A highly conserved serine or threonine residue in the conserved region 1 (CR1) region (Ser1216 in UNC-73), a stretch of hydrophobic leucine residues in the conserved region 3 (CR3) region (LLLKELL in Dbl), and the C-terminal Gln-Glu residues of the last ␣-helix of Dbl homology (DH) domain (Asn673Asp674 in Dbl), have been proposed to be important determinants involved in the GTPase interaction because mutations of these residues in UNC-73, Trio, or Dbl have led to an apparent loss of guanine-nucleotide exchange factors (GEFs) activity toward Rac1 or Cdc42 [13, 14]
To test the in vitro GEF activity, each DH mutant was expressed in Sf9 insect cells as glutathione S-transferase (GST) fusion and was purified to homogeneity by glutathione affinity chromatography (Fig. 2A)
Summary
Construction of Mutant Dbl cDNA—The DH domain point mutants were generated by oligonucleotide-directed mutagenesis of human oncogenic Dbl cDNA in pBluescript vector by the polymerase chain reaction-based second extension amplification technique using the Pfu polymerase (Stratagene), with primers that contained the desired mutations [16]. 5 g of each GST-fused small G-proteins were immobolized on agarose-glutathione beads, washed three times in a buffer containing 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 2 mM EDTA, and mixed with the precleaned cell lysates (ϳ200 g of total protein) for 1 h. Cos-7 cells were grown to ϳ80% confluence in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum before transfection with 0.1 g of pKH3-Cdc or pKH3-RhoA cDNA with or without 0.5 g of pKH3-Dbl using the LipofectAMINE regent (Life Technologies, Inc.). Transformation Assay—The cDNAs encoding the DH-PH module of wild type Dbl and DH mutants (residues 498 – 825) were subcloned into the mammalian expression vector pZipneo at the BamHI site with an N-terminal GST fusion.
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