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.     

A regulated secretory pathway in cultured hippocampal astrocytes.

Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40-50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca(2+), a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca(2+)](i) transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca(2+)](i) increase upon specific stimuli is present in cultured hippocampal astrocytes.     

ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.     

Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes

Trophic deprivation contributes to astrocyte damage that occurs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to novel cytoprotective strategies. Cultured mouse astrocytes responded to trophic deprivation with a large and transient increase in the expression of p21^ras, which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2',7'-dichlorofluorescein diacetate. The increase in p21^ras levels was largely attenuated by the reducing agent, N -acetylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha-Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha-Ras without changing Ha-Ras mRNA levels. The increase in Ha-Ras levels was sensitive to the protein synthesis inhibitor, cycloheximide, suggesting that serum deprivation increases translation of preformed Ha-Ras mRNA. The late decline in Ha-Ras levels observed after 60 min was prevented by the proteasome inhibitor, MG132, as well as by the selective mitogen-activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cultured astrocytes, as shown by an increase in phosphorylated extracellular signal-regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock-down of Ha-Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an increased expression in Ha-Ras, which is limited by the concomitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.     

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H(2)O(2))-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H(2)O(2) stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H(2)O(2) on the PLD response was mimicked by menadione-induced production of endogenous H(2)O(2). Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H(2)O(2) was totally suppressed by chelation of both extracellular and cytosolic Ca(2+) with EGTA and BAPTA/AM, respectively. Furthermore, H(2)O(2)-induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H(2)O(2) was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H(2)O(2) also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.     

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.     

High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts

Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence.     

Mutant p21ras in vinyl chloride exposed workers

The production of mutations in cellular oncogenes such as ras is involved in the development of many human cancers. These mutations result in the expression of mutant forms of the encoded p21 protein which can potentially serve as a biomarker for this carcinogenic process. Workers exposed to vinyl chloride (VC) who are at risk for the development of the sentinel neoplasm angiosarcoma of the liver (ASL) represent a model population for the study of such a mutant p21ras biomarker, since VC is known to cause a specific ras mutation in ASL. In order to determine the relationship between VC exposure and this biomarker, serum samples from a cohort of 225 French VC workers and 111 age-sex-race-smoking-drinking matched unexposed controls were examined for the presence of mutant p21ras by immunoblotting with a mouse monoclonal antibody specific for the mutant protein. Stratifying the exposed workers by degree of VC exposure in estimated ppm-years by quartiles yielded a statistically significant trend for increasing odds ratio for sero-positivity of the p21ras biomarker with increasing exposure. These results suggest that this serum biomarker is related to VC exposure and may be an early indicator of carcinogenic risk in exposed individuals.     

Mutant p21ras in vinyl chloride exposed workers

The production of mutations in cellular oncogenes such as ras is involved in the development of many human cancers. These mutations result in the expression of mutant forms of the encoded p21 protein which can potentially serve as a biomarker for this carcinogenic process. Workers exposed to vinyl chloride (VC) who are at risk for the development of the sentinel neoplasm angiosarcoma of the liver (ASL) represent a model population for the study of such a mutant p21ras biomarker, since VC is known to cause a specific ras mutation in ASL. In order to determine the relationship between VC exposure and this biomarker, serum samples from a cohort of 225 French VC workers and 111 age-sex-race-smoking-drinking matched unexposed controls were examined for the presence of mutant p21ras by immunoblotting with a mouse monoclonal antibody specific for the mutant protein. Stratifying the exposed workers by degree of VC exposure in estimated ppm-years by quartiles yielded a statistically significant trend for increasing odds ratio for sero-positivity of the p21ras biomarker with increasing exposure. These results suggest that this serum biomarker is related to VC exposure and may be an early indicator of carcinogenic risk in exposed individuals.     

A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase

Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.     

Somatostatin alters beta-adrenergic receptor-effector coupling in cultured rat astrocytes.

The neuropeptide somatostatin potentiates beta-adrenergic receptor-mediated cAMP formation in astrocytes derived from neonatal rat cortex but does not affect cAMP levels by itself. beta-Adrenergic receptors in these cells can be specifically labeled with the high affinity antagonist [125I] cyanopindolol ([125I]CYP). In addition, astrocytes display both high and low affinity binding sites for the agonist isoproterenol, which are thought to represent receptors which are coupled or uncoupled, respectively, to the guanine nucleotide regulatory protein. We find that somatostatin does not modify beta-receptor density, nor receptor affinity for either the antagonist ([125I]CYP) or for the agonist isoproterenol. In the presence of the guanine nucleotide analogue, Gpp(NH)p, only low affinity (uncoupled) displacement of [125I]CYP binding by isoproterenol is observed. However, somatostatin (1 microM), when added to the cells together with Gpp(NH)p, prevents the nucleotide-induced loss of the high affinity (coupled) component of agonist displacement. This result suggests that somatostatin increases noradrenaline-induced cAMP production by enhancing coupling between the beta-receptor and the stimulatory guanine nucleotide regulatory protein.     

Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver.

We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21-like small GTP-binding protein (24KG) which was purified from the microsomes-Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0-kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart.     

An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.     

On the mechanism of guanosine triphosphate hydrolysis in ras p21 proteins.

The residue Gln61 is assumed to play a major role in the mechanism of ras p21, and mutations of this residue are often found in human tumors. Such mutations lead to a major reduction in the rate of GTP hydrolysis by the complex of ras p21 and the GTPase activating protein (GAP) and lock the protein in a growth-promoting state. This work examines the role of Gln61 in ras p21 by using computer simulation approaches to correlate the structure and energetics of this system. Free energy perturbation calculations and simpler electrostatic considerations demonstrate that Gln61 is unlikely to serve as the general base in the intrinsic GAP-independent reaction of p21. Glutamine is already a very weak base in water, and surprisingly the GlnH+ OH-reaction intermediate is even less stable in the protein active site than in the corresponding reaction in water. The electrostatic field of Glu63, which could in principle stabilize the protonated Gln61, is found to be largely shielded by the surrounding solvent. However, it is still possible that Gln61 is a general base in the GAP/ras p21 complex since this system could enhance the electrostatic effect of Glu63. It is also possible that the gamma-phosphate acts as general base and that Gln61 accelerates the reaction by stabilizing the OH- nucleophile. If such a mechanism is operative, then GAP may enhance the effect of Gln61 by preorienting its hydrogen bonds in the transition-state configuration.     

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.