Bacterially synthesized c-Ha-ras protein (Ras) was incubated with guanosine triphosphatase (GTPase) activating (GA) protein in the presence of various phospholipids. The stimulation of Ras GTPase activity by GA protein was inhibited in some cases. Among the lipids most active in blocking GA protein activity were lipids that show altered metabolism during mitogenic stimulation. These included phosphatidic acid (containing arachidonic acid), phosphatidylinositol phosphates, and arachidonic acid. Other lipids, including phosphatidic acid with long, saturated side chains, diacylglycerols, and many other common phospholipids, were unable to alter GA protein activity. The interaction of lipids with GA protein might be important in the regulation of Ras activity during mitogenic stimulation.

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.

A cytoplasmic protein has been identified that inhibits the guanosine triphosphatase (GTPase) activity of bacterially synthesized, cellular H-Ras protein. This GTPase inhibiting protein is able to counteract the activity of GTPase activating protein (GAP), which has been postulated to function as a negative regulator of Ras activity. The potential biological importance of the GTPase inhibiting protein is further supported by its interaction with lipids. Phospholipids produced in cells as a consequence of mitogenic stimulation increase the activity of the GTPase inhibiting protein, as well as inhibit the activity of GAP. The interaction of such lipids with each of these two regulatory proteins would, therefore, tend to increase the biological activity of Ras and stimulate cell proliferation.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS‐cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.

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....

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...

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced.One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.Among the earliest and most dramatic biochemical changes induced by mitogenic stimulation is the metabolism of several types of lipid (9,10,13,19,21). Diacylglycerol, phosphatidic acid, arachidonic acid, and metabolites of phosphotidylinositol are among the lipids whose metabolism is most rapidly altered by mitogenic stimuli. The role of these alterations in the mitogenic process is unclear but likely is of critical importance. Consistent with the potential importance of lipid metabolism in mitogenic stimulation are the observations that several proto-oncogenes are functionally related to enzymes able to alter lipid metabolism. For example, growth factor receptor and related tyrosine kinasecontaining oncogenes associate with (8, 11, 12), phosphorylate (14, 15, 18, 24, 29), or bear sequence homologies to (23) phospholipase C-y and phosphatidylinositol-3-kinase. The experiments described here were designed to provide additional evidence for the participation of lipids in the control of proliferation by identifying lipids produced at the time of mitogenic stimulation that were able to affect cellular ras activity.The proto-oncogene ras is considered to play a central role in the control of proliferation in many different cell types. Not only is this gene mutated in numerous naturally occurring tumors (4), but microinjection experiments involving neutralizing anti-ras antibodies (17, 22) or two separate trans-dominant ras-inhibitory mutant proteins (D. W. Stacey, J. B. Gibbs, and L. A. Feig, unpublished data) confirm that cellular ras activity is required for the initiation of a cycle of DNA synthesis in numerous cell types. The control of cellular ras activity is not well understood, but it is clear that stimulation of ras activity involves an increase in the proportion of ras protein bound to GTP. For this reason, the GTPase-activating protein (GAP) of ras is likely to play a critical role in the regulation of ras activity. GAP can apparently funct...

In the experiments described above, a neutralizing anti-ras antibody was utilized to study the role of ras protein in normal cell proliferation. Initially, it was demonstrated that the antibody was specific for ras protein, and that ras activity was efficiently inhibited. With the neutralizing antibody, it was first shown that ras activity is required for the proliferation of all normal cell types tested. ras activity was required just prior to initiation of S phase. The transforming activity of several retroviral oncogenes was also blocked following anti-ras injection. This included the tyrosine kinase, plasma-membrane-associated proteins, and an oncogene derived from a growth factor. On the other hand, cytoplasmic oncogenes with serine kinase activity were not dependent on ras activity for expression of the transformed phenotype. These observations form the basis of our model for proliferative signal transduction. We propose that the action of either growth factors, their receptor molecules, or related oncogenes initiate an intracellular signal received by ras proteins and then transferred by ras to cytoplasmic serine kinase oncogenes. This signal transduction system directly regulates cellular proliferation. Although further evidence in support of this model is needed, it appears from our studies that the mechanism of signaling between tyrosine kinases and ras proteins might be at the level of phospholipid metabolism. This observation is based on the fact that the mitogenic lipid molecules tested were remarkably dependent on ras activity, even more so than the growth factors or related oncogenes tested. Finally, our work suggests a fundamental distinction between normal and tumor cells. All the normal cell types tested were efficiently inhibited in proliferation by the injected antibody. Tumor cells, on the other hand, were never completely inhibited by the antibody and often were not inhibited at all. The presence of an activated ras oncogene within the tumor assured at least a partial role for ras activity in the proliferation of the mature tumor line. The significance of the observed distinction between normal and tumor cells is not known. The fact that this distinction involves a protein with an apparently critical role in normal proliferation suggests that the observation might be important.