A Cytoplasmic Protein Inhibits the GTPase Activity of H-Ras in a Phospholipid-Dependent Manner

Tsai, Men-Hwei; Yu, Chun-Li; Stacey, Dennis W.

doi:10.1126/science.2237442

Cited by 153 publications

(56 citation statements)

References 21 publications

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“…Lipids and lipid metabolites such as PA, polyphosphoinositides, and arachidonic acid strongly inhibit the activity of the NF1 protein and, to a lesser extent, that of pl20GAP (4,35). There are also reports of a GTPase inhibitory protein that blocks the action of GAPs on p2lras and can be activated by DAGs and the lipids that inhibit GAPs (34). These lipids are unlikely to be important in PKC regulation of p2lras, because there is no evidence in T lymphocytes that PKC induces increases in cellular levels of lipid metabolites such as arachidonic acid or PA.…”

Section: Discussionmentioning

confidence: 98%

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.

Izquierdo¹,

Downward²,

Graves³

et al. 1992

Mol. Cell. Biol.

143

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T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p2l1 in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p2l'as, and it has been proposed that the TCR effect on p21r' occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p2l', a permeabilized cell system was used to examine TCR regulation of p2l1 under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p2l1 activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p2l'-GTP complexes, and this stimulation correlated with an inactivation of p2l' GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKCmediated, TCR-induced stimulation of p2l'. These data indicate that two mechanisms for p21l'S regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p2l1 via a non-PKC-controlled route that may involve tyrosine kinases.The three ras proto-oncogenes, Ha-, Ki-, and N-ras, encode 21,000-molecular-weight guanine nucleotide-binding proteins that cause cell transformation when constitutively activated by point mutation (3). The activity of p2lras is normally regulated by a cycle of binding GTP to give the biologically active form of the protein followed by hydrolysis of bound GTP to GDP (22). The GDP-bound form of p2lras is inactive and is reactivated by exchange of bound GDP for free cytosolic GTP. Recent studies have shown that in T lymphocytes, triggering of the T-cell antigen receptor (TCR), the adhesion molecule CD2, or the interleukin 2 (IL-2) receptor caused a very rapid stimulation of p2lras, as measured by its conversion from the GDP-to the GTP-bound state in activated cells (9,11,12). Ras proteins can also be regulated in fibroblasts by signals generated by the receptors for platelet-derived growth factor, epidermal growth factor, and insulin (5,29,30). In mast cells, p2lras is activated by the cytokines IL-3 and granulocyte macrophage colony-stimulating factor (31).In T lymphocytes, phorbol esters that activate protein kinase C (PKC) can mimic TCR triggering and induce accumulation of p21ras-GTP complexes (9). The TCR is known to stimulate PKC by a mechanism involving the generation of diacylglycerols (DAGs; the endogenous PKC activators) via phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids (PtdIns) (17,27). TCR-stimulated PKC could thus mediate the p2lras activation seen upon triggering of this receptor. The proposal that PKC functions as an upstream regulator of p2lras in T cells is in contrast to the previously described link between p2lras and PKC in fibroblasts, where, based on observations that "active" * Corresponding author.

show abstract

Section: Discussionmentioning

confidence: 98%

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.

Izquierdo¹,

Downward²,

Graves³

et al. 1992

Mol. Cell. Biol.

143

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show abstract

“…In this regard, the generation of phosphatidic acid may be important. It has been suggested that PA or more likely a metabolite of PA inhibits the H-Ras GTPase inhibitory protein, GAP (40,44). Thus, the production of PA might enhance v-Src signals going through H-Ras, which has previously been implicated in v-Src signaling (36).…”

Section: Discussionmentioning

confidence: 99%

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

1991

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Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with araehidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG pkroduction and. enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.Protein kinase C (PKC) activity has been implicated in v-Src-induced intracellular signals (5,12,26,33,35,37,43). Diacylglycerol (DAG) is a second messenger that leads to the activation of PKC (25). DAG is generated from phospholipids either directly, through the action of type C phospholipases, or indirectly, through the action of type D phospholipases that generate phosphatidic acid (PA), which is then converted to DAG by a PA phosphatase (reviewed by Exton [10]). Increased levels of DAG have been observed in cells transformed by v-Src (22, 43); however, the source of the increased DAG is unclear. The time course for increased inositol phosphate levels in v-Src-transformed cells did not correlate with the time course for increased DAG (15,22), suggesting that DAG produced in response to v-Src is generated from a source other than phosphatidylinositol (P1) or phosphorylated derivatives of PI. Over the last several years, considerable data have been presented implicating PI hydrolysis by type C phospholipases as the source of increased DAG levels in response to a variety of stimuli (reviewed by Berridge [2]). Although it is likely that type C phospholipase-mediated hydrolysis of PI constitutes a major signaling pathway leading to the production of biologically active inositol phosphates, it is not clear that increases in DAG levels seen in response to a variety stimuli can be accounted for by hydrolysis of this relatively minor phospholipid (10). In this regard, it has recently been demonstrated that phosphatidylcholine (PC) rather than PI is the major source of increased DAG production in response to a variety of stimuli (la, 3, 6, 14, 19, 20, 23, 30, 32, 42 then washed with isotonic Tris-saline buffer, rapidly treated with 0.6 ml of methanol (MeOH)-6 N HCl (50:2), and scraped from the culture dish. The MeOH-HCl-treated cells were then extracted with 0.6 ml of CHCl3. Phase separation was obtained by adding 200 ,ul of 1 M NaCl. The organic phase was recovered, dried under N2, ...

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“…These lipid affinity columns have also been utilized to identify a second protein with the ability to alter the GTPase rate of Ras proteins (29 (3,16,21,22). Serum has been reported to stimulate the production of arachidonic acid in different cell types (8-12, 14, 15, 23).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1991

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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...

show abstract

A Cytoplasmic Protein Inhibits the GTPase Activity of H-Ras in a Phospholipid-Dependent Manner

Cited by 153 publications

References 21 publications

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

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

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