Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25Mm originate both loss-of-function and gain-of-function proteins.
The role of two residues within the catalytic domain of CDC25Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25Mm(E1048K) and CDC25Mm(S1122V) were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25Mm(S1122A) showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.
Carrera
V
V
Dipartimento di Fisiologia e Biochimica Generali Sezione di Biochímica Comparata, Università degli Studi di Milano, Milan, Italy.
Moroni
A
A
Martegani
E
E
Volponi
C
C
Cool
R H
RH
Alberghina
L
L
Vanoni
M
M
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D016762:Genes, fos; D006150:Guanine Nucleotides / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D006150:Guanine Nucleotides; D020666:ras-GRF1; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins