REGULATED AND CONSTITUTIVE ACTIVITY BY CDC25(MM) (GRF), A RAS-SPECIFIC EXCHANGE FACTOR

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV of apparently full-length cDNAs from a single mammalian gene, ca lled CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain spe cific, with the other three classes, although they have distinct 5' en ds, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an incr ease in the basal level of GTP . Ras. Serum stimulation of these trans formants lead to a further increase in GTP . Ras only in cells express ing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimula te guanine nucleotide release from GDP . Ras in vitro. When NIH 3T3 ce lls and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras p rotein and the other encoding a cytosolic Ras protein, the basal level s of GTP bound to both forms of the mutant Ras protein were significan tly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ra s protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchang e on Ras, the presence of N-terminal sequences is associated with a se rum-dependent change in GTP . Ras, and the serum-dependent increase in GTP . Ras by exogenous CDC25Mm or by endogenous exchange factors prob ably requires membrane association of both Ras and the exchange factor .