Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm).
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.
Vanoni
M
M
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza, 2 20126 Milano, Italy.
Bertini
R
R
Sacco
E
E
Fontanella
L
L
Rieppi
M
M
Colombo
S
S
Martegani
E
E
Carrera
V
V
Moroni
A
A
Bizzarri
C
C
Sabbatini
V
V
Cattozzo
M
M
Colagrande
A
A
Alberghina
L
L
D000818:Animals; D002460:Cell Line; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009154:Mutation; D051381:Rats; D015398:Signal Transduction / Q000235:genetics; D020666:ras-GRF1 / Q000235:genetics* / Q000378:metabolism
D020666:ras-GRF1