THE MINIMAL ACTIVE DOMAIN OF THE MOUSE RAS EXCHANGE FACTOR CDC25(MM)

The minimal active domain of the mouse CDC25(Mm), 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 Pas proteins in vitro. A C-terminal domain of 256 residues (CDC25(1005-1260)(Mm)) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25(1005-1233)(Mm)) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25(1030-1260)(Mm) 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 CDC25(1005-1260)(Mm), 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 CDC25(974-1260)(Mm). Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25(Mm) is shorter than the yeast one. (C) 1995 Academic Press. Inc.