No 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...

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.

Summary Meningiomas are common, mostly benign, tumours arising from leptomeningeal cells of the meninges, which frequently contain mutations in the neurofibromatosis type 2 (NF2) gene. In this study, we analysed a protein product of the neurofibromatosis type 1 (NF1) gene, neurofibromin, in human established leptomeningeal cells LTAg2B, in 17 sporadic meningiomas and in a meningioma from a patient affected by NF2. The expression level of neurofibromin was determined by immunoblotting and immunoprecipitation with anti-neurofibromin antibodies. The functional status of neurofibromin was analysed through its ability to stimulate the intrinsic GTPase activity of p21 ras. In the cytosolic extracts of four sporadic meningiomas and in the NF2-related meningioma, the expression level and the GTPase stimulatory activity of neurofibromin were drastically reduced compared with the level present in the human brain, human established leptomeningeal cells LTAg2B and the remaining 13 meningiomas. Our results suggest that neurofibromin is expressed in leptomeningeal cells LTAg2B and in most meningiomas, i.e. tumours derived from these cells. The reduced expression and GTPase stimulatory activity of neurofibromin was found in about 23% of meningiomas and in the single NF2-related meningioma analysed. These results suggest that decreased levels of neurofibromin in these tumours may contribute to their tumorigenesis.