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Mutations at position 1122 in the catalytic domain of the mouse ras‐specific guanine nucleotide exchange factor CDC25 Mm originate both loss‐of‐function and gain‐of‐function proteins
Author(s) -
Carrera Vittorio,
Moroni Andrea,
Martegani Enzo,
Volponi Celina,
Cool Robbert H.,
Alberghina Lilia,
Vai Marco
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01481-1
Subject(s) - mutant , complementation , guanine nucleotide exchange factor , protein fragment complementation assay , saccharomyces cerevisiae , mutagenesis , guanine , yeast , biochemistry , biology , nucleotide , mutation , chemistry , microbiology and biotechnology , gene , signal transduction
The role of two residues within the catalytic domain of CDC25 Mm , 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 CDC25 and CDC25 were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25 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.