Ras GTPase-activating protein physically associates with mitogenically active phospholipids.

Abstract

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.Cellular Ras proteins play an important role in controlling cellular proliferation by acting as a component of the proliferative signal transduction pathway (2,20). Recently, a cytoplasmic protein was identified which stimulates more than 100-fold the GTPase activity of normal Ras but not that of its oncogenic mutant (27). Since the GTPase-activating protein (GAP) can convert biologically active Ras-GTP into the inactive Ras-GDP complex, GAP may be a negative regulator of Ras protein (34). On the other hand, other analyses indicate that GAP might be a Ras effector protein (1,5,24). In either case, both GAP activity and Ras activity are likely to be critical in the control of cellular proliferation.On the basis of microinjection studies, we previously reported that the biological activity of Ras might be controlled by phospholipids (32). Consistent with this hypothesis, GAP activity (and hence the nucleotide status of Ras) was found to be inhibited by certain lipids (30). Lipids whose metabolism is altered during mitogenic stimulation (e.g., phosphatidic acid [PA], phosphatidylinositol phosphates, and arachidonic acid) were most active in blocking GAP activity, while more abundant lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were totally inactive. Furthermore, in studies of mitogen-stimulated NIH 3T3 cells, a lipid was identified which had the ability to inhibit GAP activity. This lipid was produced within 3 min of mitogen stimulation but only in subconfluent cells (33). These biochemical studies, therefore, support the suggestion that GAP might be inhibited by certain lipids.The production of lipids able to inhibit GAP activity might represent a novel mechanism for regulating the activity not only of Ras but also of ras-related genes. To test this possibility, the R-ras and Rho proteins (which have considerable biochemical and sequence homology with Ras) were analyzed. As with Ras, it was found that each of these related proteins failed to be stimulated by their respective GAP in the presence of lipids which are sim...

A Requirement for Calcium-independent Phospholipase A2 in Thrombin-induced Arachidonic Acid Release and Growth in Vascular Smooth Muscle Cells

“…Because CREB/ ATF-1 mediate cAMP-response element-dependent gene expression, one possible mechanism by which p38 MAPK is involved in thrombin-induced growth in VSMC is via activation of iPLA 2 , thereby releasing arachidonic acid, which in turn stimulates ATF-1-dependent gene expression. Previous work from other laboratories showed that arachidonic acid and its eicosanoid metabolites are involved in the mediation of mitogenic signaling events such as formation of focal adhesions (43)(44)(45)(46). Because both cPLA 2 and iPLA 2 appear to be involved in agonist-induced arachidonic acid release (11, 20, 23-25, 29, 30) and a role for both PLA 2 s has been demonstrated in the regulation of cell growth (27,28,31), it would be interesting to learn which of these PLA 2 s is involved in receptor tyrosine kinase and G protein-coupled receptor agonist-induced focal adhesion formations.…”

5(S)-Hydroxyeicosatetraenoic Acid Stimulates DNA Synthesis in Human Microvascular Endothelial Cells via Activation of Jak/STAT and Phosphatidylinositol 3-Kinase/Akt Signaling, Leading to Induction of Expression of Basic Fibroblast Growth Factor 2

“…5-LOX products of arachidonic acid such as leukotriene C 4 have also been shown to mediate epidermal growth factor-induced cytoskeletal rearrangement (9). The findings that eicosanoids, particularly the LOX products of arachidonic acid, mediate mitogen-induced signaling events via inhibition of GTPase activating proteins offer additional support for their role in cell growth (11,12,49). In addition to their role in the mitogenic signaling events cited above, some eicosanoids such as prostaglandin F 2␣ and 12(R)-hydroxy-5,8,14-eicosatrienoic acid induce the expression of angiogenic factors bFGF-2 and VEGF in rat aortic smooth muscle cells and rabbit limbal microvessel endothelial cells, respectively (50,51).…”

“…Arachidonic acid and its metabolites, known as eicosanoids, are involved in the regulation of a variety of biological processes including vascular tone (5,6). In addition, these lipid molecules have been reported to mediate intracellular signaling events in response to a number of stimuli (7)(8)(9)(10)(11)(12). A large body of pharmacological data suggests that arachidonic acid and its eicosanoid metabolites stimulate mitogenesis in various cell types, including cancer cells (13)(14)(15)(16)(17)(18)(19)(20).…”

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

“…There are numerous reports of PA effects on protein kinases such as the PKC (138,173,280), protein kinase N, and p21 cdcrac families (126,129,167,267,281). PA also appears to have inhibitory effects on Ras-GTPase-activating proteins (252) and Rho-GTP-dissociating isoforms (37; reviewed in Refs. 19,115,126,155).…”