Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins.

Abstract

We have previously identified a membrane factor capable of stimulating guanine nucleotide exchange activity for ras p21 proteins. The ras guanine nucleotide exchange factor (rGEF) was purified from bovine brain to near homogeneity by successive chromatographies on DE52 DEAEcellulose, Sepharose 6B, hydroxylapatite, and FPLC phenylSuperose resins. SDS/polyacrylamide gel electrophoresis of the purified rGEF showed a single major protein with a molecular mass of 35 kDa. rGEF increased the exchange rate of GDP in normal [Gly 21p21 or oncogenic [VaIl2Jp21 up to 30-to 40-fold under physiological concentrations of Mg2e. Since the factor was free from GDP/GTP binding activity and nonspecific GDP hydrolytic activity, we propose that rGEF may regulate GDP/GTP exchange reaction of ras proteins in response to external growth signals. Moreover, rGEF enhanced the dissociation of bound GDP from some of ras-like G proteins, R-ras, rapl-A, rabl-B, and rho proteins, raising the possibility that rGEF may affect the activities of these proteins.Mammalian ras p21 proteins are regarded as one of the regulatory components in the metabolic pathway related to cell proliferation and cell transformation. Normal p21 proteins, such as guanine nucleotide-binding regulatory (G) proteins, bind GDP and GTP and have an intrinsic GTPase activity (1). Although it is assumed that, in response to a cell proliferation signal, p21 exchanges bound GDP for GTP and that GTP-p21 activates its effector molecule, the precise mechanism of the ras regulation remains unclear. A cytoplasmic protein, GTPase activating protein (GAP), has been identified (2, 3) that accelerates the GTP hydrolysis in normal p21 but not in oncogenic p21. GAP could, as an upstream signal terminator, convert active GTP-p21 to inactive GDP-p21 through the hydrolysis of GTP bound to the p21 or act as a downstream effector, similar to the translation elongation factor EF-Tu (4). The later model is favored by the fact that GAP cannot induce the hydrolysis of GTP bound to mutant p21 proteins, which fail to interact with their effector.However, some observations raise the possibility that the guanine nucleotide exchange reaction is also an important event for p21 function. The GDP-GTP exchange reaction of p21 was inhibited by Y13-259 ras-neutralizing antibody, which also blocked the induction of cellular DNA synthesis (5, 6). In yeast Saccharomyces cerevisiae cells, the CDC25 gene product was postulated to catalyze, either directly or indirectly, the GDP-GTP exchange reaction for the RAS2 protein, which is structurally related to mammalian ras proteins (7). Furthermore, the proliferation of NIH 3T3 cells was inhibited by the [Asp17]p21 mutant with preferential affinity for GDP. This down-regulation could come from the competition between mutant and normal p21 proteins for coupling with the regulatory cellular components that catalyze the nucleotide exchange reaction (8).We have identified (9) a membrane factor that markedly enhanced the exchange of GDP bound to Ha-ras p21 proteins...

Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2

“…Fig. 3 is a series of GTP/GDP exchange experiments, the hallmark of GEF function (11)(12)(13)(14)(15). Fig.…”

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

“…Effector domain peptides have, in fact, been shown to inhibit transport in the biosynthetic pathway (Plutner et al, 1990). Proteins have been identified that can escort rab proteins in the cytosol (GDI), stimulate GTP hydrolysis or facilitate GDP/GTP exchange (Huang et al, 1990;Sasaki et al, 1990;West et al, 1990;Burstein et al, 1991;Tan et al, 1991;Burstein and Macara, 1992). Other proteins, which include putative components of the transport machinery, are expected to interact with rab proteins.…”

An interaction between p21ras and heat shock protein hsp60, a chaperonin.

“…In recent years, a number of proteins have been identified that are implicated in direct interactions with p21tm' in yeast and in mammals (2,11). In mammals, the proteins interacting with p21tm' may be placed into two groups: those that potentiate the GTPase activity of p21tm5, such as GTPase-activating protein (GAP) and the locus involved in hereditary neurofibromatosis (NF-1) (12)(13)(14) and those that catalyze GTP/GDP exchange by p21tm' (15)(16)(17)(18). Nevertheless, their role in the ras pathway is still unclear (2,9,19).…”