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 (CDC25 Mm1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25 Mm1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm1030-1260 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 CDC25Mm1005-1260, 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 CDC25Mm974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one. © 1995 Academic Press, Inc.
Coccetti, P., Mauri, I., Alberghina, L., Martegani, E., Parmeggiani, A. (1995). The minimal active domain of the mouse Ras exchange factor CDC25Mm. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 206(1), 253-259 [10.1006/bbrc.1995.1035].
The minimal active domain of the mouse Ras exchange factor CDC25Mm
COCCETTI, PAOLA;ALBERGHINA, LILIA;MARTEGANI, ENZO;
1995
Abstract
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 (CDC25 Mm1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25 Mm1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm1030-1260 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 CDC25Mm1005-1260, 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 CDC25Mm974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one. © 1995 Academic Press, Inc.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.