The GTPase-activating NF1 fragment of 91 amino acids reverses v-Ha-Ras-induced malignant phenotype.

The human neurofibromatosis type 1 gene encodes a Ras GAP (GTPase-activating protein) of 2818 amino acids called NF1. This GAP contains a domain of 338 amino acids (residues 1194-1531) called NF1-GRD, which shares 26% sequence identity with the C-terminal domain (GAP1C, residues 709-1044) of another Ras GAP called GAP1. Both NF1-GRD and GAP1C activate normal Ras GTPases but not oncogenic mutants such as v-Ha-Ras. Any attempt to reverse the v-Ha-Ras-induced malignant transformation by the GAP1C has, so far, been unsuccessful. However, we have found that when the NF1-GRD is overexpressed in v-Ha-Ras-transformed NIH/3T3 cells, it greatly reduces their ability to form colonies in a soft agar, the property tightly associated with their malignancy. This is, so far, the first demonstration that a Ras-binding protein can act as a potent antagonist of the oncogenic Ras mutants in mammalian cells. In an attempt to further screen the smallest anti-oncogenic fragment derived from the NF1-GRD, we have prepared a series of its deletion mutants and examined their interaction with Ras first by monitoring their GAP activity (ability to activate the normal Ras GTPase). The deletion analysis has revealed that the N-terminal 247 amino acids (residues 1194-1440) of NF1-GRD are not required for its GAP activity, suggesting that its remaining domain of 91 amino acids (NF91, residues 1441-1531) is sufficient to bind the v-Ha-Ras, although its GAP activity is 20 times lower than the NF1-GRD. The NF91 is indeed able to reverse the v-Ha-Ras-induced malignant transformation as the NF1-GRD. The NF91 is, so far, the smallest among the tumor suppressor proteins that show the anti-v-Ha-Ras action in vivo. Thus, the NF91 appears to be a good starting material from which a smaller and more potent v-Ha-Ras antagonist could be devised to be used as a potential cure for the human cancers caused by the Ras mutants.