Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25 originate both loss-of-function and gain-of-function proteins |
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Authors: | Vittorio Carrera Andrea Moroni Enzo Martegani Celina Volponi Robbert H Cool Lilia Alberghina Marco Vanoni |
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Institution: | aDipartimento di Fisiologia e Biochimica Generali Sezione di Biochimica Comparata, Università degli Studi di Milano, via Celoria, 26, 20133 Milan, Italy;bMax-Planck-Institute für Molekulare Physiologie, Postfach 102664, 44026 Dortmund, Germany |
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Abstract: | 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 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. |
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Keywords: | Guanine nucleotide exchange factor Site-directed mutagenesis fos-Luciferase Oncogene BIAcore Yeast |
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