Molecular Biology of GAP and its Interaction with Oncogenic ras p21

Abstract

The interaction of ras p21 with guanine nucleotides is central to its biological function. Mutations that impair the intrinsic GTPase or that enhance GTP for GDP exchange, activate ras cell-transforming activity. Both of these types of mutations result in an increased level of ras p21-GTP complex. By analogy with G-protein regulatory cycles therefore, the ras p21-GTP complex would be predicted to be the biologically active species. This prediction appears to be true for both the mammalian and yeast systems. Discrepancies between the in vitro GTPase activities and biological potencies which were observed, prompted the testing of the model through the quantitation of the GTP and GDP nucleotides bound to both normal and oncogenic ras proteins in vivo. These measurements were first made for ras proteins expressed in yeast cells where the ras proteins could be overproduced. Yeast RAS1 and RAS2 proteins, overexpressed in [32P] orthophosphate-labelled cultures of S. cerevisiae cells, were found to be bound almost entirely to GDP, whereas increased amounts of GTP were bound to RAS proteins containing oncogenic mutations that impair GTPase activity. Unexpectedly, normal mammalian ras p21 was bound to near equimolar proportions of both GTP and GDP nucleotides, whereas the oncogenic forms were bound almost exclusively to GTP (Gibbs et al., 1987).