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Abstract
Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF(W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21(ras), and strongly reduce affinity of the nucleotide-free ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.
Highlights
Ras proteins are highly conserved guanine nucleotide proteins, endowed with intrinsic low GTPase activity [1]
CDC25MmW1056E and CDC25MmT1184E in Vitro Induce Noncatalytic Dissociation of Ras-bound Nucleotides—One of our laboratories has been carrying out studies on structure/function relationships on a ras-specific guanine nucleotide exchange factors (GEF), CDC25Mm, by sitedirected mutagenesis [44]
In this paper we report that tryptophan 1056, which is universally conserved in all ras-specific GEF, is essential for GEF activity
Summary
Recombinant and Genetic Manipulations—Recombinant DNA manipulations were performed according to standard methods [34]. CDC25Mm PCR from A10 Genomic DNA and CDC25Mm RT-PCR— Genomic DNA was prepared from several clones of A10 rat embryonic aortic smooth muscle cells transfected with recombinant pcDNA3 plasmid containing the mutant CDC25MmW1056E gene according to published protocols [39]. The binary complex ras1⁄7CDC25Mm was made by incubating a 5-fold molar excess of p21ras1⁄7GDP in 50 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 5 mM dithioerythritol buffer containing 10 mM EDTA with wild-type or mutant GST-GEF (pre-bound to glutathione-Sepharose) for 30 min at room temperature. The labeled complexes were prepared by incubating 1 M p21ras protein in buffer A (50 mM Tris-Cl, pH7.5, 1 mM MgCl2, 100 mM NH4Cl, 1 mg/ml bovine serum albumin) for 10 min at 30 °C with 3 mM EDTA and 20 M [3H]GDP (10.1 Ci mmolϪ1, Amersham Pharmacia Biotech). For determination of noncatalytic dissociation rates, GEF concentration was raised up to 30-fold, i.e. at a 3:1 molar ratio with p21ras
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