The minimal active domain of the mouse ras exchange factor CDC25Mm.
The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.
Coccetti
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P
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Mauri
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I
Alberghina
L
L
Martegani
E
E
Parmeggiani
A
A
D000818:Animals; D018797:Cell Cycle Proteins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D016283:Proto-Oncogene Proteins p21(ras)