Nerve and Epidermal Growth Factor Induce Protein Synthesis and eIF2B Activation in PC12 Cells (original) (raw)
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FEBS Letters, 2002
In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)-stimulated extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre-treatment with apigenin or LY294002 sustained EGF-stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor-induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF-induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin. ß
… and cellular biology, 1997
Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of ⌬Raf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by ⌬Raf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp ؊974 and ؊988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following ⌬Raf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to ⌬Raf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following ⌬Raf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.
PC12-E2 cells: A stable variant with altered responses to growth factor stimulation
Journal of Cellular Physiology, 1995
A variant cell line, designated E2, characterized by more rapid responses to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) and markedly more robust responses to interleukin-6 and 8-Br-cAMP, has been subcloned from the rat PC12 cell line. The enhanced responsiveness to NGF in E2 cells is not due to receptor overexpression as judged by TrkA protein levels and tyrosine kinase activity, but may be associated with the increased and prolonged tyrosine phosphorylntion of ERKl (extracellular signal regulated kinase 1) and ERK2. The rapid morphological differentiation induced by different growth factors in E2 cells is mediated in a transcription-independent manner suggesting that E2 cells may constitutively express some differentiation-associated molecules that allow direct entry into the neuronal program. o 1995 WiIey-Liss, Inr.
Journal of Biological Chemistry, 2003
We have previously demonstrated that insulin-like growth factor 1 (IGF1) induces eukaryotic initiation factor 2B (eIF2B) activation in neuronal cells through the phosphatidylinositol 3 kinase/glycogen synthase kinase 3 pathway, as well as by activation of the MAPK-activating kinase (MEK)/mitogen-activated protein kinase (MAPK) signaling pathway (Quevedo, C., Alcazar, A., and Salinas, M. (2000) J. Biol. Chem. 275, 19192-19197). This paper addresses the mechanism involved in IGF1-induced eIF2B activation via the MEK/MAPK cascade in cultured neurons treated with IGF1, and demonstrates that extracellular signal-regulated MAP kinase 1 and 2 (ERK1 and 2) immunoprecipitates of IGF1-treated neuronal cells promote this activation. This effect did not directly result from eIF2B phosphorylation by ERK immunoprecipitates. In addition, recombinant ERK1 and 2 neither activate eIF2B nor phosphorylate it. Endogenous protein phosphatase 1 and 2A catalytic subunits (PP1C and PP2AC respectively) were co-immunoprecipitated with ERK1 and 2, and the association of ERK with PP1C was stimulated by IGF1 treatment, resulting in increased PP1 activity. ERK immunoprecipitates incubated with PP1 inhibitors did not activate eIF2B, indicating that PP1C activates eIF2B. In vitro experiments with phosphorylated eIF2B showed that recombinant PP1C (α isoform) dephosphorylates and activates eIF2B. Paralleling eIF2B activation, IGF1 treatment induced PP1 activation in a MEK/MAPK-dependent fashion. Moreover, the treatment of neurons with the PP1 inhibitor tautomycin inhibited PP1 activation and prevented IGF1-induced eIF2B activation. These findings strongly suggest that IGF1-induced eIF2B activation in neurons is effected by PP1, the activation of which is mediated by the MEK/MAPK signaling pathway. by guest on August 4, 2016 http://www.jbc.org/ Downloaded from The translational inhibition caused by eIF2B inhibition through eIF2α phosphorylation is a well-known cellular mechanism that triggers in response to different stress situations (18-20). However, in growth factor-treated cells, and in response to other different treatments, by guest on August 4, 2016 http://www.jbc.org/ Downloaded from EXPERIMENTAL PROCEDURES Materials-IGF1, inhibitor 2 (I2), tautomycin, purified recombinant PP1 catalytic subunit (PP1C) α isoform (PP1Cα), anti-ERK1 and 2 polyclonal antibody, and anti-diphospho-ERK1 and 2(Thr 183 and Tyr 185 in ERK2) (the active forms of the kinases) monoclonal antibodies, were provided by Sigma. Purified recombinant PP1C γ isoform (PP1Cγ), 4EBP1 and fostriecin were from Calbiochem, purified recombinant ERK1 and GSK3β, and anti-PP2A catalytic subunit (PP2AC) polyclonal antibody were purchased from Upstate Biotechnology, and purified recombinant ERK2 and anti-phospho-GSK3α/β(Ser 21 /Ser 9 ) (the inactive form of the kinases) polyclonal antibodies were provided by New England Biolabs. PD98059 was obtained from Biomol, LY294002 from Alexis, anti-ERK2 and anti-eIF2α polyclonal antibodies from Santa Cruz Biotech, and anti-PP1C and anti-GSK3β monoclonal antibodies from Transduction Laboratories. Leibovitz L-15, Ham's F-12 and high glucose Dulbecco's media were purchased from Life Technologies. [ 3 H]GDP and [γ-32 P]ATP were supplied by Amersham Biosciences, and synthetic peptides by Mimotopes. eIF2 and eIF2B were purified from calf brain (38).
Biochemical Journal, 1996
The transforming growth factor β (TGF-β) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-β type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immunoprecipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-β1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and...
Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells
Journal of Cellular Biochemistry, 1982
Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A43 1 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains -50% and clone 4 contains -20 % of the EGF-stimulated protein kinase activity of the parental A43 1 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:l mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.
Molecular and Cellular Biology, 1997
Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP ...