Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins.

Abstract

Certain lipids were found to inhibit the interaction between rho and R-ras proteins and their respective GTPase-activating proteins (GAP). Inhibitory lipids were similar for each protein but differed significantly from those previously found to inhibit the interaction between ras protein and GAP activity. These data raise the possibility that ras and related proteins are controlled biologically by interactions between lipids and GAP molecules.R-ras and rho are related to the proto-oncogene ras by sequence homology as well as by their ability to bind and hydrolyze GTP (11,12). Ras is part of a signal transduction mechanism leading to cellular proliferation (14), but no function is known for either R-ras or rho. In contrast to control of classical G proteins involved in signal transduction (8), the biological and biochemical control of ras and ras-related proteins is poorly understood. A GTPase-activating protein (GAP) which is a potential negative regulator of ras activity has been characterized (16), but because ras-GAP remains active in proliferating cells its potential role in the control of ras (and therefore cellular proliferation) is unclear (10, 16). Recently, however, certain lipids whose metabolism is known to be altered in mitogenically stimulated cells were shown to disrupt the effect of ras-GAP upon purified ras protein (17). It is now clear that ras-GAP interacts with R-ras, whereas the rho protein interacts with a distinct smaller molecule, rho-GAP (6). In this study, a similar but nonidentical group of lipids is shown to disrupt the interaction between R-ras and GAP and between rho and rho-GAP. The biological significance of these observations is not yet clear, but the data raise the possibility that lipids might normally function to control the activity of ras-related proteins through interactions with GAP molecules.The ras-related proteins R-ras and rho were purified from a bacterial expression system (6), and their GTPase activities were assayed by measuring the loss of radiolabeled _y32p from protein-bound GTP. The GTPase activities were relatively slow for rho and R-ras proteins in the absence of added GAP activity as previously observed for ras protein.Addition of GAP activity as a crude cytoplasmic extract increased the GTPase rate severalfold (Fig. 1). The presence of a 29-kilodalton protein with GAP activity specific for rho has been demonstrated in such extracts, along with the 125-kilodalton ras-GAP activity (6, 7). Previous studies indicate that the ability of ras-GAP to stimulate GTPase activity of ras protein was inhibited by certain lipids (17). We were anxious to test the possibility that lipids might also inhibit the GAP activity associated with other ras-related proteins. For this analysis, a variety of lipids were coincubated with rho protein and GAP-containing cell lysates. Certain of the lipids tested were able to inhibit the ability of rho-GAP to stimulate GTPase activity of rho protein (Table 1). In particular, 60 ,ug of added phosphatidic acid per ml * Corresponding author....

Beta-actin mRNA localization is regulated by signal transduction mechanisms.

“…One reported effect of PDGF and other growth factors is activation of ras-GTP (Gibbs et al, 1990;Satoh et al, 1990), which then initiates either cell division through activation of raf (Moodie et al, 1993) or chemotaxis through activation of rac and rho (as seen by increased actin polymerization, focal adhesions and stress fibers) . LPA can also induce activation of the (Tsai et al, 1989). The ability of both LPA and PDGF to induce/3-actin mRNA localization, suggests that this localization may possibly be through the and rac as well.…”

“…LPA is a potent stimulator of platelet aggregation which has been shown to activate the rho-class of GTPases (Tsai et al, 1989;. Addition of LPA to serum-starved CEFs resuited in a significant increase in ceils exhibiting peripheral ~-actin mRNA (Fig.…”

Elevated levels and mitogenic activity of lysophosphatidylinositol in k‐ras‐transformed epithelial cells

“…GTPase-activating-protein-(GAP)-induced stimulation of ras-p21 GTPase activity was assayed as previously described [23]. ras-p21 (kindly donated by Dr A.…”

Arachidonic acid activates Jun N-terminal kinase in vascular smooth muscle cells

“…In addition, several small GTP binding proteins such as Rac and Cdc42 Coso et al, 1995;Minden et al, 1995) and protein tyrosine kinases such as Pyk2 and Vav Tokiwa et al, 1996) have been reported to stimulate JNK/SAPK pathway and lie upstream to the MEKK1. In fact, earlier studies have demonstrated that arachidonic acid and its LOX metabolites, HETEs, activate these GTP binding proteins by inhibiting the activity of their respective GTPase activating proteins (GAPs) (Han et al, 1991;Yu et al, 1990;Tsai et al, 1988Tsai et al, , 1989. Because arachidonic acid and its LOX metabolites possess the ability to activate GTP-binding proteins such as Rac, it is likely that these proteins play a role in the activation of JNK1 by these lipid molecules.…”