Biochemical characterization of Artemia ras p21

The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze GP₄G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.     

Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis.

Tumor cell lines derived from malignant schwannomas removed from patients with neurofibromatosis type 1 (NF1) have been examined for the level of expression of NF1 protein. All three NF1 lines examined expressed lower levels of NF1 protein than control cells, and the level in one line was barely detectable. The tumor lines expressed normal levels of p120GAP and p21ras. Although the p21ras proteins isolated from the tumor cells had normal (nonmutant) biochemical properties in vitro, they displayed elevated levels of bound GTP in vivo. The level of total cellular GAP-like activity was reduced in extracts from the tumor line that expresses very little NF1 protein. Introduction of the catalytic region of GAP into this line resulted in morphological reversion and lower in vivo GTP binding by endogenous p21ras. These data implicate NF1 protein as a tumor suppressor gene product that negatively regulates p21ras and define a "positive" growth role for ras activity in NF1 malignancies.     

Erythropoietin induces p21ras activation and p120GAP tyrosine phosphorylation in human erythroleukemia cells.

Erythropoietin is the major regulator of the proliferation and differentiation of erythroid precursors, but little is known about its molecular mechanism of action. Using a human erythroleukemic cell line (HEL), we investigated whether p21ras is involved in erythropoietin signal transduction. We found that stimulation of HEL cells with erythropoietin induces a 5-fold increase in the amount of GTP bound to the endogenous p21ras. This effect is dose-dependent and occurs very rapidly. We also observed that erythropoietin causes tyrosine phosphorylation of several proteins in a time-dependent manner that correlates with the p21ras activation. Moreover, inhibition of tyrosine kinases by genistein totally prevents the erythropoietin-induced accumulation of a p21ras.GTP complex. By using an antiserum against the GTPase-activating protein, we found that p120GAP is rapidly phosphorylated in tyrosine in response to erythropoietin. Furthermore, the ability of a lysate from erythropoietin-stimulated HEL cells to induce in vitro hydrolysis of GTP bound to p21ras was strongly reduced. These results demonstrate that activation of p21ras is an early event in the erythropoietin signal transduction pathway, and they suggest that accumulation of the p21ras.GTP complex may be triggered by inhibition of GTPase-activating protein activity.     

Activation of p21ras by transforming growth factor beta in epithelial cells.

The transforming growth factor beta (TGF beta) family members are ubiquitously expressed and control a variety of cellular processes by interacting with at least two types of high affinity cell surface receptors. However, the primary signal transduction mechanism of the receptors is unknown. The ras-encoded 21-kDa GTP binding proteins have recently been shown to mediate the effects of other polypeptide growth factors. Here we show that both TGF beta 1 and TGF beta 2 (5 ng/ml) result in a rapid (within 6 or 12 min, respectively) stimulation of GTP bound to p21ras in TGF beta-sensitive intestinal epithelial cells. Further, the CCL64 epithelial cell line, extremely sensitive to growth inhibition by TGF beta, displayed a concentration-dependent increase in GTP bound to p21ras by TGF beta 1 and a rapid activation of p21ras by TGF beta 2. The results provide the first direct evidence for rapid activation of a receptor coupling component for TGF beta in epithelial cells.     

Normal cellular Ha ras p21 protein causes local disruption of bilayer phospholipid

We have investigated the interactions of the p21 protein of c-Ha ras with its phospholipid environment. Gel filtration of detergent-"solubilized" p21 revealed that this preparation consisted of a mixture of multimolecular aggregates of protein and phospholipid and also a population of individual p21 molecules. Addition of 8 M urea to p21 preparations increased the solubility of the molecule in detergent solutions upon the removal of this denaturant. The progressive addition of the detergent cholate appeared to increase the efficiency of p21 preparations to bind GTP. This affinity for GTP was not removed even at high detergent concentrations, when delipification of the p21 was presumably effected. Modification of the composition of the phospholipid species surrounding the protein did not appear to alter its affinity for GTP. Electron spin resonance studies with membrane spin-labels indicated a perturbation of the bilayer extending to between 44 and 100 phospholipids surrounding the molecule. However, no evidence was found for any population of intimately bound phospholipid, which is seen as an annulus of about 30 lipids in transmembrane proteins such as Ca2+-ATPase. From these results we propose that the Ha ras p21 protein has the ability to associate directly with the membrane in a manner clearly discernible from that of a transmembrane protein.     

Regulation of p21ras activity.

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.     

Activation of cellular p21ras by myristoylation

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.     

Oligomeric structure of p21 ras proteins as determined by radiation inactivation

Using radiation inactivation we determined that p21 ras proteins exhibit an oligomeric target size when assayed both structurally and functionally. Similar target sizes of p21 in ras-transformed cells and in purified preparations of the protein suggested that its structure is homo-oligomeric. p21 monomers were destroyed by radiation with the same target size as the GTP binding activity, indicating the occurrence of a tight association allowing energy transfer between the monomers. Irradiation in the presence of GTP, dithiothreitol, or EDTA did not change the target size. Normal (Gly12) and transforming (Lys12) forms of the protein exhibited similar target sizes. The homo-oligomeric structure suggests that p21 ras proteins do not conform to the structure of monomeric alpha subunits in classical G proteins (alpha beta gamma heterotrimers) and establishes similarities with other homo-oligomeric proteins (such as Escherichia coli CRP) which acquire the active conformation through subunit reorientation upon nucleotide binding.     

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.     

p21ras mediates control of IL-2 gene promoter function in T cell activation.

It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin-2 (IL-2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL-2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL-2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL-2. Furthermore, using a dominant negative mutant of ras, Ha-rasN17, we show that endogenous ras function is essential for induction of IL-2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL-2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.     

Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D.

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.     

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.     

Association of p21ras with cellular polypeptides

p21ras specific antiserum was used to immunoprecipitate p21ras polypeptides from human A431 cells. In addition to p21ras, this antiserum precipitated a series of polypeptides with relative molecular weights of 150,000, 120,000, 105,000, and 50,000. The precipitation of these polypeptides was prevented by preincubation of the antiserum with an excess of purified Ras protein. These polypeptides do not share an epitope with p21ras, and two of them (120 and 150 kDa) copurify with a fraction of p21ras. The co-precipitation of p21ras with these polypeptides was detected in a variety of cell types. The pattern of the immunoprecipitates was consistently different in normal and ras-transformed cells. The 120- and 150-kDa polypeptides are phosphorylated on serine and threonine in A431 cells. Serum treatment resulted in a 2-fold increase in the phosphoserine content of the 120-kDa polypeptides.     

NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation.

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.     

How does p21ras transform cells?

Oncogenic forms of p21ras are found in a wide range of human tumors. However, the mechanism by which p21ras transforms remains obscure. Genetic evidence has identified a domain of p21ras that is involved with interaction with an effector molecule required for transformation. Two proteins, GAP and the tumor suppressor NF1, interact with p21ras in this region but it is an unresolved puzzle whether either of these is the an unresolved puzzle whether either of these is the effector. After interaction with an effector, two downstream events--activation of protein kinase C and another pathway--are necessary for induction of DNA synthesis by oncogenic p21ras; however, morphological transformation does not require activation of protein kinase C.     

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.     

Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

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

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras 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 p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.