A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras.
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TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Here we report that oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. Our results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells. (+info)
Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae.
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The TOR (target of rapamycin) signal transduction pathway is an important mechanism by which cell growth is controlled in all eucaryotic cells. Specifically, TOR signaling adjusts the protein biosynthetic capacity of cells according to nutrient availability. In mammalian cells, one branch of this pathway controls general translational initiation, whereas a separate branch specifically regulates the translation of ribosomal protein (r-protein) mRNAs. In Saccharomyces cerevisiae, the TOR pathway similarly regulates general translational initiation, but its specific role in the synthesis of ribosomal components is not well understood. Here we demonstrate that in yeast control of ribosome biosynthesis by the TOR pathway is surprisingly complex. In addition to general effects on translational initiation, TOR exerts drastic control over r-protein gene transcription as well as the synthesis and subsequent processing of 35S precursor rRNA. We also find that TOR signaling is a prerequisite for the induction of r-protein gene transcription that occurs in response to improved nutrient conditions. This induction has been shown previously to involve both the Ras-adenylate cyclase as well as the fermentable growth medium-induced pathways, and our results therefore suggest that these three pathways may be intimately linked. (+info)
Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway.
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We studied the role of Ras signaling in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, caused excessive cell death in developing eyes at pupal stages. The Argos-induced cell death was suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppressed the Argos-induced cell death. Thus, Argos-induced cell death seemed to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid was suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death. (+info)
Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.
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The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle. (+info)
Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas.
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Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis. (+info)
Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation.
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Accumulating evidence indicates that the interdomain B regions of ZAP-70 and Syk play pivotal roles in the coupling of T-cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP-70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP-70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP-70, the resulting ZAP(Y319-->F) mutant failed to reconstitute TCR-dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT-dependent transcription in ZAP-70-deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)-gamma1 and the LAT adapter protein in the ZAP(Y319-->F)-expressing cells. On the other hand, ZAP(Y319-->F)-expressing cells displayed normal increases in SLP-76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP-70 with the SH2 domains of two key signaling molecules, Lck and PLC-gamma1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP-70-containing signaling complex that leads to the activation of the PLC-gamma1- and Ras-dependent signaling cascades in antigen-stimulated T cells. (+info)
Modulation of Ras/Raf/extracellular signal-regulated kinase pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells.
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Endothelial cells (ECs) exposed to cyclic strain induce gene expression. To elucidate the signaling mechanisms involved, we studied the effects of cyclic strain on ECs by using early growth response-1 (Egr-1) as a target gene. Cyclic strain induced a transient increase of Egr-1 mRNA levels that resulted in an increase of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. ECs subjected to strain enhanced Egr-1 transcription as revealed by promoter activities. Catalase pretreatment inhibited this induction. ECs, transfected with a dominant positive mutant of Ras (RasL61), increased Egr-1 promoter activities. In contrast, transfection with a dominant negative mutant of Ras (RasN17) attenuated this strain inducibility. ECs transfected with a dominant negative mutant of Raf-1 (Raf301) or the catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK2) diminished strain-induced promoter activities. However, little effect on strain inducibility was observed in ECs transfected with a dominant negative mutant of Rac (RacN17) or a catalytically inactive mutant of JNK (JNK[K-R]). Consistently, strain-induced Egr-1 expression was inhibited after ECs were treated with a specific inhibitor (PD98059) to mitogen-activated protein kinase kinase. Moreover, strain to ECs induced mitogen-activated protein kinase/ERK activity. The activation of the ERK pathway was further substantiated by an increase of strain-induced transcriptional activity of Elk1, an ERK substrate. This strain-induced ERK activity was attenuated after ECs were treated with N-acetylcysteine or catalase. Consequently, this Egr-1 gene induction was abolished after ECs were treated with N-acetylcysteine or catalase. Deletion analyses of the promoter region (-698 bp) indicated that cyclic strain and H2O2 shared a common serum response element. Our data clearly indicate that cyclic strain-induced Egr-1 expression is mediated mainly via the Ras/Raf-1/ERK pathway and that strain-induced reactive oxygen species can modulate Egr-1 expression at least partially via this signaling pathway. (+info)
Control of the cell morphology and the S phase entry by mitogen-activated protein kinase kinase. A regulatory role of its n-terminal region.
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The mitogen-activated protein kinase kinase (MAPKK)/MAP kinase (MAPK) cascade plays an important role in the growth control of mammalian cells. We have found that expression of constitutively active MAPKK induces rapid morphological changes of fibroblastic cells, which are accompanied by disruption of stress fibers and disappearance of focal adhesions. These changes took place under the conditions that inhibited cellular Ras function, suggesting a linkage between the MAPK cascade and the control of cell morphology. We further show that constitutively active MAPKK can induce expression of endogenous Fos protein, an immediately early gene product, and cause the S phase entry of G0-arrested cells. Finally, expression of the N-terminal fragment of MAPKK which encompasses the nuclear export signal sequence and the MAPK-binding site blocked both the serum-induced S phase entry of quiescent cells and the oncogenic Ras-induced morphological changes. All these results demonstrate that MAPKK is one of key molecules involved in the control of both cell morphology and cell proliferation and suggest an important role for the N-terminal region of MAPKK in the regulation of the MAPK signaling. (+info)