Oncogene studies have involved ras encoded proteins, which have been analyzed by examining proteins that influence the activity of Ras protein. Schwannoma cell lines from patients with neurofibromatosis had low levels of the NFl product neurofibromin, which correlated with their containing high levels of GTP.Ras. These results were consistent with NFl being a tumor suppressor gene whose encoded GTPase stimulation negatively regulates Ras. We have also identified neuroblastoma and melanoma cell lines with genetic abnormalities of NF1 and reduced to absent levels of neurofibromin, suggesting that NF1 is acting as a tumor suppressor gene in these cell lines. In contrast to the schwannoma lines, the level of GTP.Ras was low in all lines and did not correlate with that of neurofibromin. These results suggested that NF1 might inhibit cell growth by a mechanism independent of its GTPase stimulatory activity. To confirm this hypothesis, a full length NF1 cDNA was introduced into NIH 3T3 cells. The cells that overexpressed neurofibromin grow more slowly and had normal levels of GTP.Ras. Introduction of the NF1 cDNA into melanoma lines slowed their growth and induced a differentiated phenotype, including an increase in cell size, dendrite formation, and an increase in tyrosinase. We have also identified four types (I-IV) of apparently full-length cDNAs from a gene CDC25Mm that encodes a ras-specific exchange factor. All four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Analysis of expressing ras mutants in these cells indicated that the serum-dependent increase in GTP.Ras by CDC25Mm or by endogenous exchange factors requires membrane association of both Ras and the exchange factor. Morphological transformation of NIH 3T3 cells was observed following co-expression the amino terminus of GAP (GAP- N) v-src (MDSRC) lacking the membrane-localizing sequence. Further analysis suggested that tyrosine phosphorylation and complex formation involving GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.
