Identification of a nucleotide exchange-promoting activity for p21ras.

Abstract

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21w has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchangepromoting protein acts reversibly and does not cause degradation of p211. It is inactive towards the a subunit of a heterotrimeric GTP-binding protein (G..) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchangepromoting), has the characteristics expected of a physiological activator of p21w in cellular growth-signal-transduction pathways.Members of the ras family of oncogenes encode Mr 21,000 proteins (p21) that bind and hydrolyze guanine nucleotides. The transforming potential of the ras protooncogenes can be activated by point mutations that either decrease the intrinsic GTPase activity of p21 or increase the rate with which p21 exchanges guanine nucleotides (1). Activated ras oncogenes have been found in up to 30% of human tumors (2). By analogy with other oncogene products, it has been speculated that p21 normally functions as a component of a growthsignal-transduction pathway. One would therefore expect it to interact with several distinct cellular proteins that act variously to impart signals to p21, to receive signals from p21, or to modulate signal transduction. A protein that activates the GTPase activity of p21 (GAP) has been identified (3); this may represent a downstream effector ofp21 action (4, 5). The nature of the upstream elements of the pathway that control p21 activity remains unknown: by analogy with the heterotrimeric GTP-binding proteins (G proteins) (6) it might be expected that stimulation of the activity of p21 would involve an increase of the rate at which it exchanges guanine nucleotide with soluble pools. Here we report the identification of an activity in a human tissue extract that promotes the exchange of guanine nucleotides on p21. This factor, termed REP (ras exchange-promoting), has properties consistent with its being a physiological activator of the ras proteins.MATERIALS AND METHODS Purification and Assay of Exchange Activity. Two normal human placentas were homogenized in a domestic blender with 1 liter of ice-cold 50 mM potassium phosphate buffer (pH 7.5) containing 1 mM EGTA, 0.3 M sucrose, 1 mM dithiothreitol (DTJ7), 10 gg of soybean trypsin inhibitor per ml, 10 ,Ag of leupeptin per ml, 10 ,ug of aprotinin per ml, and 10 mM benzamidine. Particulate matter was pelleted by centrifugation at 50...

Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.

“…In the case of ras p21, GTPase-activating protein (GAP) interacts with p21-GTP and enhances the GTPase activity of ras p21 (34,35). Stimulators of the dissociation of the ras p2l-GDP complex have also been reported (36)(37)(38)(39). However, no evidence showing that these factors are responsible for altering the GDP/GTP state of ras p21 in response to exogenous stimuli has been presented.…”

FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

“…They hydrolyze GTP to GDP, and the rate of hydrolysis is regulated by GTPase activating proteins or GAPs (37). Stimulation of guanine nucleotide exchange, induced by Ras exchange factors (38), will lead to GTP loading and activation of Ras. Activation of Ras can thus be accomplished either by inhibiting GAPs or stimulating exchange factors.…”

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).…”