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1.
p21ras mediates control of IL-2 gene promoter function in T cell activation. 总被引:20,自引:0,他引:20
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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. 相似文献
2.
J D Graves J Downward S Rayter P Warne A L Tutt M Glennie D A Cantrell 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(11):3709-3712
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. 相似文献
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4.
Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. 总被引:10,自引:3,他引:10
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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. 相似文献
5.
Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.
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C J Molloy T P Fleming D P Bottaro A Cuadrado S A Aaronson 《Molecular and cellular biology》1992,12(9):3903-3909
Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling. 相似文献
6.
EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease. 相似文献
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The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. 总被引:5,自引:3,他引:2
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p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation. 相似文献
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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. 相似文献
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12.
We have previously demonstrated that the low number of interleukin-4 receptors (IL-4Rc) on HL-60 leukemia cells render this population susceptible to differentiation by IL-4. As it occurs with normal human monocytes, IL-4 induces the expression of HLA-DR surface antigens on HL-60 cells as well. The second messenger pathway(s) involved after the IL-4 stimulation leading to class II up-regulation has not been fully examined. Here we show that IL-4-induced class II antigen expression on the HL-60 cell line or normal human monocytes is calcium/calmodulin-independent since theophylline (TPH, a calmodulin inhibitor) does not block the IL-4 effect. In addition, the pyruvate kinase C (PKC) pathway does not seem to participate in the process either because in our system activation of PKC by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) is insufficient by itself to induce HLA-DR. We found, however, that a second messenger pathway can be mediated by a G protein system since IL-4 concomitantly induces class II and p21ras expression which can be successfully blocked by a highly specific anti-p21ras monoclonal antibody. In addition, using another p21ras inducer, the 5-azacytidine C (5-AzaC), we showed that this agent can also induce the expression of p21ras and class II, both of which can be inhibited by the same antibody. Thus, it appears that IL-4 selects the G protein system as a signaling pathway in order to exert its action for the induction of HLA-DR on human normal monocytes or M2 leukemia target cells. Since monocytes and macrophages participate in virtually all immune reactions, the regulation of class II induction is of obvious importance. 相似文献
13.
Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C. 总被引:2,自引:1,他引:1
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The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C. 相似文献
14.
Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function. 总被引:10,自引:5,他引:5
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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. 相似文献
15.
Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. 总被引:50,自引:28,他引:22
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We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response. 相似文献
16.
Y Lu L Raptis S Anderson M J Corbley Y C Zhou H Pross T Haliotis 《Biochimie et biologie cellulaire》1992,70(10-11):1249-1257
The positive association of the ras oncogene with human cancer and the recognition that malignancy may, in part, represent the imbalance between cell proliferation and differentiation have generated intense interest in the potential role of ras in cell differentiation. We investigated this possibility utilizing as a model system the differentiation of the mesenchymal cell line C3H 10T1/2 (10T1/2) to adipocytes, and a series of transfectants of 10T1/2 cells in which the level of the ras gene product (p21ras; Ras) can be effectively up- or down-modulated. In agreement with previous reports, we found that 10T1/2 cultures, propagated in the resting state for several weeks, spontaneously convert to fat cells at a very low frequency. Downmodulation of endogenous p21ras levels, as a consequence of expression of antisense ras, markedly increased the rapidity and frequency of adipose conversion (6- to 10-fold), which was equivalent in magnitude to that effected by the potent differentiating agent 5-azacytidine. Conversely, overexpression of ras completely inhibited cell differentiation. In addition, adipocytes derived from antisense-ras expressing lines were characterized by a decrease in hormone responsiveness, as well as an apparent deficiency in attaining the terminally differentiated state. These findings suggest that Ras may be a negative regulator of the decision-making step of fibroblast differentiation to adipocytes. In addition, Ras may play an essential positive role in the transduction of hormonal signals necessary for full adipocyte maturation during later progression along the differentiation pathway. 相似文献
17.
Regulatory mechanisms for ras proteins. 总被引:12,自引:0,他引:12
J Downward 《BioEssays : news and reviews in molecular, cellular and developmental biology》1992,14(3):177-184
The proteins encoded by the ras proto-oncogenes play critical roles in normal cellular growth, differentiation and development in addition to their potential for malignant transformation. Several proteins that are involved in the control of the activity of p21ras have now been characterised. p120GAP stimulates the GTPase activity of p21ras and hence acts as a negative regulator of ras proteins. It may be controlled by tyrosine phosphorylation or association with tyrosine phosphorylated proteins. The neurofibromatosis type 1 (NF 1) gene also encodes a potential GTPase activating protein which is likely to be subject to a different control mechanism. Guanosine nucleotide exchange factors for p21ras have now been identified: these may be positive regulators of ras protein function. It appears that p21ras is subject to rapid regulation by several distinct mechanisms which are likely to vary in different cell types; the ras proteins are thereby able to act as very sensitive cellular monitors of the extracellular environment. 相似文献
18.
Involvement of p21ras distinguishes positive and negative selection in thymocytes. 总被引:20,自引:4,他引:16
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K A Swan J Alberola-Ila J A Gross M W Appleby K A Forbush J F Thomas R M Perlmutter 《The EMBO journal》1995,14(2):276-285
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. 相似文献
19.
Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines 总被引:2,自引:0,他引:2
D L Jankovic M Gibert D Baran J Ohara W E Paul J Theze 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(9):3113-3120
We have investigated the effects of IL-2 and IL-4 on different parameters of T cell activation using three T cell lines. The Th cell line L14 and the cytotoxic T cell line C30.1, both grown in IL-2-containing medium, and a line derived from C30.1 cells (line 1) cultured in IL-4 for a prolonged period were studied. All three cell lines could be activated with IL-2 or IL-4. T cell stimulation by either IL-2- or IL-4-induced identical patterns of cell size enlargement and transferrin receptor expression. However, only IL-2 up-regulated cell-surface expression of the p55 subunit of the IL-2R (p55 IL-2R) as measured by flow cytometry and RIA. This difference was also reflected by the accumulation of soluble p55 IL-2R in the culture medium. No significant increase in expression of membrane or soluble forms of p55 IL-2R was detected after IL-4 stimulation. mAb specific for p55 IL-2R which block IL-2-induced T cell growth did not affect IL-4-mediated T cell proliferation indicating that p55 IL-2R is not involved in IL-4-mediated T cell growth. Analysis of IL-4R expression performed on line 1 using biotinylated IL-4 revealed that IL-4, but not IL-2, is capable of increasing IL-4R expression. Together these results suggest that during IL-2- or IL-4-induced T cell proliferation, each lymphokine specifically up-regulates its own receptor. 相似文献
20.
cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. 总被引:33,自引:2,他引:31
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In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action. 相似文献