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Address correspondence to Xiang-Xi Xu, Ovarian Cancer and Tumor Cell Biology Programs, Dept. of Medical Oncology, Medical Science Division, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: (215) 728-2188. Fax: (215) 728-2741. email: X_Xu{at}fccc.edu
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Abstract |
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Key Words: retinoic acid; c-Fos; Elk-1; cytoskeleton; nucleocytoplasmic translocation
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Introduction |
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In addition to changes in cellular morphology and gene expression, the differentiated F9 cells exhibit a suppressed or impaired response to growth factors or serum and a reduced growth rate (Sherman and Miller, 1978; Faria et al., 1999). In many mammalian cell types, the RasMAPK cascade is the principal mitogenic signaling pathway and MAPK activation is essential for cell growth (Brunet et al., 1999; Hochholdinger et al., 1999; Kim-Kaneyama et al., 2000). The RasMAPK signaling pathway mediates the cellular response to multiple growth factor receptor tyrosine kinases, and the transmittance of the signal from the extracellular receptor at the cell surface to changes in gene expression has been well described (Robinson and Cobb, 1997; Chang and Karin, 2001). Specifically, a linear pathway from receptor tyrosine kinaseRasRafMAPK kinase (MEK) results in phosphorylation and activation of MAPK or extracellular signalregulated kinase (ERK), p44/42 MAPK or ERK1 and 2, respectively, by MEK and subsequent phosphorylation/activation of, among several targets, the transcription factor Elk-1. Elk-1 is responsible for transcriptional activation of the immediate early gene c-fos through binding to the Ets/SRE element in the c-fos promoter (Gille et al., 1992; Marais et al., 1993; Yang et al., 1999). c-Fos interacts with the transcription factor Jun to form the AP-1 complex, which mediates the biological response, including cell cycle progression in serum-starved growth-arrested cells (Field et al., 1992). Moreover, c-Fos expression contributes to and is required for the malignant growth of solid tumors (Angel and Karin, 1991; Saez et al., 1995; Arteaga and Holt, 1996), and down-regulation of c-Fos expression interferes with the growth of tumor cells in vitro (Arteaga and Holt, 1996). Thus, c-Fos is likely a site of regulation in cell growth control (Altin et al., 1992; Brown et al., 1998; Vanhoutte et al., 2001). In F9 cells treated for 4 d with RA to induce endodermal differentiation, serum causes a rapid and significant activation of MAPK; however, c-Fos expression is consistently suppressed (Smith et al., 2001a,b). This uncoupling of MAPK activation from c-Fos expression occurs at the step of Elk-1 phosphorylation/activation by MAPK.
Both the duration and the localization of the Ras/MAPK signal are normally regulated during proliferation and differentiation of many cell types (Pouyssegur et al., 2002). Dual phosphorylation of MAPK on tyrosine and threonine by MEK occurs in the cytoplasm, and several nonspecific phosphoserine/phosphothreonine- and phosphotyrosine-specific phosphatases and a MAPK-specific phosphatase (MKP3) have been reported to dephosphorylate and inactivate p44/p42 MAPK/Erk (Camps et al., 1998; Keyse, 2000), effectively terminating the signal. Activated MAPK must translocate into the nucleus to phosphorylate Elk-1 and other nuclear targets. The MAPK-specific phosphatases MKP1 and MKP2, which are neosynthesized in response to MAPK pathway stimulation (Volmat et al., 2001), are also stabilized by MAPK-dependent phosphorylation (Brondello et al., 1999) and reside in the nucleus (Brondello et al., 1995), where they may also rapidly terminate MAPK activity acting in a feedback loop. Presumably, under resting conditions, nonphosphorylated MAPK is complexed with MEK in the cytoplasm, and upon phosphorylation disassociates from MEK and either freely diffuses as a monomer through nuclear pores (Adachi et al., 1999), homodimerizes and enters the nucleus via a carrier-free/nuclear poreindependent mechanism (Khokhlatchev et al., 1998), or interacts with the nuclear pore complex for entry (Matsubayashi et al., 2001; Whitehurst et al., 2002). In the nucleus, the signal must be terminated by dephosphorylation and MAPK relocated to the cytoplasm via a MEK-dependent active transport (Adachi et al., 2000).
To understand how endoderm differentiation of F9 EC cells altered growth factorstimulated c-Fos expression, we focused on active MAPK and its sustained nucleocytoplasmic localization. Here, we report that in differentiated F9 EC cells, and to a similar extent in differentiated mouse ES cells, MAPK does not enter the nucleus upon serum stimulation but remains activated in the cytoplasm. Thus, in differentiated cells, the transcriptional-dependent (nuclear) and -independent (cytoplasmic) MAPK activation are uncoupled by the restriction of MAPK nuclear entry.
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Results |
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In another experiment, the fractionated cellular components were further analyzed for additional proteins (Fig. 4 C). AKT is mainly cytoplasmic. The nucleocytoplasmic distribution of p90RSK phosphorylated either dually at Thr 359/Ser 363 or on Ser 380 correlates well with the distribution of phosphorylated MAPK. In undifferentiated F9 cells, there is a small fraction of phospho-p90RSK in the nucleus, which is absent in differentiated cells. Thus, it appears that p90RSK has a separate larger nuclear and smaller cytoplasmic pool. These results can be explained by the assumption that MAPK is no longer able to enter the nucleus to phosphorylate nuclear p90RSK in differentiated cells, and only cytoplasmic p90RSK is phosphorylated.
MAPK nucleocytoplasmic location determined by immunofluorescence microscopy
To confirm that RA-induced differentiation alters MAPK nucleocytoplasmic localization, we examined activated MAPK (pErk) localization using indirect immunofluorescence confocal microscopy. In undifferentiated F9 cells, nuclear localized pErk was detectable within 5 min, reached maximum by 15 min, and remained visible 3060 min after serum addition (Fig. 5). This time course closely corresponds to MAPK activation in total F9 cell lysates determined by immunoblot analysis (Smith et al., 2001a,b; Smedberg et al., 2002). In RA-treated cells, activated MAPK could be readily detected 5 min after serum addition, reached maximum by 15 min, and remained elevated after 3 h. However, the pattern of cellular distribution of pErk is strikingly different between differentiated and undifferentiated F9 cells. In undifferentiated cells, pErk staining exhibits a punctate pattern throughout the cell body. In contrast, in differentiated cells, pErk staining is exclusively in the outer ring of the cells, and no significant pErk was detectable in the nucleus (Fig. 5). The nuclear exclusion of the active MAPK is best illustrated by overlapping of pErk staining with propidium iodide (PI) to show the nucleus (Fig. 5).
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Discussion |
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Regulation of nuclear translocation of MAPK
Nucleocytoplasmic localization of MAPK is a pivotal point in regulation of its downstream targets, including those involved in growth regulation, differentiation, and cell function. Nuclear accumulation of p42/p44 MAPK is observed after mitogenic stimulation, which may persist for up to 6 h (Volmat et al., 2001), and is believed to be accompanied by neosynthesis of nuclear anchoring proteins. The nucleus has been proposed to act as an "anchoring and inactivating center," restricting MAPK from activation by MEK in the cytoplasm (Volmat et al., 2001; Pouyssegur et al., 2002).
Although the mechanisms regulating nucleocytoplasmic translocation of MAPK are fairly well described, our finding in F9 EC and ES cells that differentiation restricts nuclear access of activated MAPK is only the second physiological example of this kind of regulation. Human diploid fibroblasts that have undergone replicative senescence, which is characterized by a loss of proliferative capacity and impaired expression of c-Fos and other immediate early genes in response to mitogens and serum, show a reduced abundance of nuclear activated MAPK (Tresini et al., 2001). The decreased nuclear abundance of active MAPK is accompanied by decreased activation of Elk-1 and reduced c-Fos expression, but the senescent fibroblasts differ from the F9 and ES cells in that total number of MAPK molecules is increased. In the senescent fibroblasts, it was suggested that the abundant unphosphorylated MAPK molecules compete for phosphorylated MAPK for dimerization and nuclear translocation, resulting in a reduced fraction of activated MAPK in the nucleus (Tresini et al., 2001). In general, systems that are aging affected, including primary rat hepatocytes in culture, mouse T lymphocytes and macrophages, human lymphocytes, melanocytes, and fibroblasts, also exhibit aging- or senescent-associated changes in MAPK signaling (Tresini et al., 2001). We did not observe a change in the total amount of MAPK protein in RA-differentiated F9 and ES cells, but rather a reduction of total MAPK located in the nucleus. Instead, we conclude that the reduced amount of activated MAPK in differentiated F9 and ES cells is the result of an actin- and microtubule-dependent inhibitory mechanism on nuclear translocation of the activated MAPK imposed by the cells after differentiation (Fig. 9). This restriction of MAPK nuclear entry may be a general mechanism for suppression of proliferation in cell differentiation, as we have begun to observe the restriction of MAPK nuclear entry in other types of differentiated cells in tissues and primary cultures (unpublished data).
Role of cytoskeleton in regulation of MAPK nuclear entry
The regulated coupling of MAPK activation and c-Fos expression may be related to morphological changes in the cytoskeleton that accompany endodermal differentiation of F9 cells (Kurki et al., 1989; Burdsal et al., 1994), such that destabilization may reconfigure a situation similar to the undifferentiated cells. Moreover, the data reported here indicate that serum-induced MAPK activation and nuclear import in F9 cells do not depend on the microtubule and microfilament network and regulation of the cytoskeleton. Rather, disruption of the cell cytoskeleton results in an enhanced MAPK activation and c-Fos expression, and suppression of c-Fos expression by RA-induced differentiation is prevented without an organized cytoskeleton. The enhanced MAPK activation by inhibition of protein synthesis and disruption of cell cytoskeleton has been previously observed (Kyriakis et al., 1994). We conclude that the restriction of MAPK activation and nuclear entry requires an intact actin and microtubule cytoskeleton.
The mechanism for the uncoupling of MAPK activation and c-Fos expression has been investigated. The reduction in activated MAPK in the nucleus of the differentiated F9 or ES cells is not due to higher nuclear export because leptomycin B had no effect nor to an increased dephosphorylation in the nucleus because active MAPK actually remains elevated for a longer period than in control cells and appears to be severalfold more abundant, especially in ES cells. It is not known if a protein that actively binds MAPK is expressed in ES and EC cells, although one such protein, PEA-15, which has been found to retain MAPK in cytoplasm in neural cells (Formstecher et al., 2001), was not found in F9 cells induced by RA (unpublished data).
When F9 cells undergo differentiation, a striking phenotype is the rearrangement of cell cytoskeleton (Kurki et al., 1989; Burdsal et al., 1994). We favor the idea that in differentiated cells, MAPK is tethered to actin and/or microtubule filaments themselves or associates with a protein complex hinged on endocytic cargos traveling along an actin or microtubule track. The arrangement of the cytoskeleton or the directional transport of endocytic cargos may restrict the access of MAPK for nuclear entry, leading to suppression of its nuclear transcriptional-dependent activity, thus growth suppression (Fig. 9). RA may induce the expression of a set of proteins such as adaptors and regulators to allow such organization. This mechanism may underlie the importance of cell adhesion and the cytoskeleton structure in the regulation of mitogenic signaling and cell proliferation.
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Materials and methods |
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Cell culture
F9 mouse EC cells were purchased from the American Type Culture Collection and cultured on gelatin-coated tissue culture plates in DME containing 10% heat-inactivated FBS and 1x antibiotic-antimycotic solution. The plates were coated with an autoclaved 0.1% gelatin solution overnight at 4°C and washed with PBS before use. All-trans-RA (referred to as RA) was added from a 1-mM stock solution in DMSO to a final concentration of 0.1 mM, generally, 24 h after plating. Control cultures received an equal volume of DMSO vehicle, which was generally 0.01% of the final culture volume. Mouse RW-4 ES cells were maintained on a layer of irradiated murine embryonic fibroblasts or feeder layer in ES cell medium with LIF (ESGRO; Chemicon; Robertson, 1987). For experiments, the cells were trypsinized and plated on gelatinized tissue culture plates without feeder cells for 2 d in ES cell medium containing LIF, and then trypsinized and plated in the presence of 0.1 mM RA for 4 d.
Cell fractionation
F9 cells were cultured 4 d in the presence of RA or DMSO vehicle, and then incubated overnight (1824 h) in DME containing 0.1% FBS and antibiotics/antimycotics (serum-depleted medium). For serum-stimulation experiments, serum-depleted medium was removed and replaced with 1520% FBS-containing medium for various times (090 min) at 37°C, 5% CO2. At the end of the treatment, cells were washed three times with ice-cold PBS containing 0.5 mM sodium orthovanadate, scraped into hypotonic lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM NaF, and a protease inhibitor cocktail; Sigma-Aldrich), and incubated on ice for 10 min (Fazioli et al., 1993). The lysate was Dounce homogenized (40 strokes) with a tight-fitting pestle and designated the total homogenate. To obtain the nuclear fraction, the homogenate was centrifuged at 375 g for 5 min, and the pellet (nuclear fraction) was washed five times with hypotonic lysis buffer containing 0.1% NP-40 to remove membrane and cytosolic contamination and resuspended in lysis buffer containing 0.5% sodium deoxycholate, 0.1% SDS, and 0.2% NP-40. The soluble fraction was centrifuged twice at 375 g to remove nuclear contamination and was designated the cytosolic fraction. Fractions were centrifuged at 12,000 g to remove insoluble material. All procedures were performed on ice. Protein was quantitated using DCC assay (Bio-Rad Laboratories) according to the manufacturer's instructions. Immunoblotting was performed according to standard procedures, as described previously (Smith et al., 2001b).
Immunofluorescence microscopy
F9 cells were first differentiated with RA and reseeded at 104 cells/well onto gelatin-coated Permanox 4-well slide chambers (Lab-Tek). For some experiments, cells were seeded directly onto and differentiated in the slide chambers. To process for immunocytochemistry, after treatment, cells were washed two times with PBS and fixed and lysed in ice-cold methanol at -20°C for 5 min (Edwards et al., 1988), washed two times in PBS, and blocked in PBS containing 3% BSA for 1 h at RT. The primary antibody was diluted (1:200) into 3% BSA in PBS containing 0.1% Tween-20 and incubated with the slides for 1 h at 37°C in a humidified chamber (Osborn and Weber, 1982). The slides were washed three times with PBS containing 1% BSA and 0.1% Tween-20 and incubated with the appropriate secondary antibody (1:1001:200) in 3% BSA at 37°C for 1 h. Slides were washed four times with PBS, stained with PI (1:3,000 dilution of a 10-mg/ml stock in water) for 2 min at RT, and washed three more times with PBS. The slides were mounted in SlowFade or ProLong media according to the manufacturer's directions. Cells were viewed on a laser scanning confocal microscope (model 200; Bio-Rad Laboratories) using a 60x water objective, and images were deconvoluted using the Laser Sharp software (Bio-Rad Laboratories) and analyzed using Adobe Photoshop.
To determine the effect of leptomycin B on MAPK and phospho-MAPK localization, serum-starved cells were incubated with 10 nM leptomycin B for 30 min in serum-free medium and stimulated with 15% FBS for 10 min in the continued presence of leptomycin B. Cells were processed for immunocytochemistry as described in the previous paragraph. Antibody controls (minus the primary antibody) demonstrated little to no fluorescence staining, indicating the specificity of the antibodies. The Erk1 mAb (BD Biosciences) was used at 1:50 dilution, and the secondary antibody, rhodamine redconjugated goat antimouse IgG (Molecular Probes), was used at 1:300. Cell nuclei were counterstained with TOTO-3 diluted 1:10,000 in PBS for 2 min at RT.
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Acknowledgments |
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This work was supported by National Cancer Institute grants R01 CA095071, CA79716, and CA75389 from the National Institutes of Health (to X.-X. Xu).
Submitted: 3 December 2003
Accepted: 6 January 2004
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References |
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Adachi, M., M. Fukuda, and E. Nishida. 1999. Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J. 18:53475358.
Adachi, M., M. Fukuda, and E. Nishida. 2000. Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 148:849856.
Altin, J.G., R. Wetts, K.T. Riabowol, and R.A. Bradshaw. 1992. Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells. Mol. Biol. Cell. 3:323333.[Abstract]
Angel, P., and M. Karin. 1991. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta. 1072:129157.[CrossRef][Medline]
Aplin, A.E., S.A. Stewart, R.K. Assoian, and R.L. Juliano. 2001. Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1. J. Cell Biol. 153:273281.
Arteaga, C.L., and J.T. Holt. 1996. Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer zenografts in nude mice. Cancer Res. 56:10981103.[Abstract]
Arceci, R.J., A.A. King, M.C. Simon, S.H. Orkin, and D.B. Wilson. 1993. Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart. Mol. Cell. Biol. 13:22352246.[Abstract]
Bastien, J., S. Adam-Stitah, J.-L. Plassat, P. Chambon, and C. Rochette-Egly. 2002. The phosphorylation site located in the A region of retinoic X receptor is required for the antiproliferative effect of retinoic acid (RA) and the activation of RA target genes in F9 cells. J. Biol. Chem. 277:2868328689.
Boylan, J.F., and L.J. Gudas. 1991. Overexpression of the cellular retinoic acid binding protein-I (CRABP-I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells. J. Cell Biol. 112:965979.[Abstract]
Brondello, J.M., F.R. McKenzie, H. Sun, N.K. Tonks, and J. Pouyssegur. 1995. Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts. Oncogene. 10:18951904.[Medline]
Brondello, J.M., J. Pouyssegur, and F.R. McKenzie. 1999. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science. 286:25142517.
Brown, J.R., E. Nigh, R.J. Lee, H. Ye, M.A. Thompson, F. Saudou, R.G. Pestell, and M.E. Greenberg. 1998. Fos family members induce cell cycle entry by activating cyclin D1. Mol. Cell. Biol. 18:56095619.
Brunet, A., D. Roux, P. Lenormand, S. Dowd, S. Keyse, and J. Pouyssegur. 1999. Nuclear translocation of p42/p44-mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J. 18:664674.
Burdsal, C.A., M.M. Lotz, J. Miller, and D.R. McClay. 1994. Quantitative switch in integrin expression accompanies differentiation of F9 cells treated with retinoic acid. Dev. Dyn. 201:344353.[Medline]
Camps, M., A. Nichols, C. Gillieron, B. Antonsson, M. Muda, C. Chabert, U. Boschert, and S. Arkinstall. 1998. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science. 280:12621265.
Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature. 410:211218.
Cho, S.Y., S.Y. Cho, S.H. Lee, and S.S. Park. 1999. Differential expression of mouse Disabled 2 gene in retinoic acid-treated F9 embryonal carcinoma cells and early mouse embryos. Mol. Cells. 9:179184.[Medline]
Cooper, J.A. 1987. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105:14731478.[Medline]
Dean, M., R.A. Levine, and J. Campisi. 1986. c-myc regulation during retinoic acid-induced differentiation of F9 cells is posttranscriptional and associated with growth arrest. Mol. Cell. Biol. 6:518524.[Medline]
Edwards, S.A., A.Y.K. Rundell, and E.D. Adamson. 1988. Expression of c-fos antisense RNA inhibits the differentiation of F9 cells to parietal endoderm. Dev. Biol. 129:91102.[Medline]
Faria, T.N., C. Mendelsohn, P. Chambon, and L.J. Gudas. 1999. The targeted disruption of both alleles of RARß2 in F9 cells results in the loss of retinoic acid-associated growth arrest. J. Biol. Chem. 274:2678326788.
Fazioli, F., L. Minichiello, B. Matoskova, W.T. Wong, and P.P. Di Fiore. 1993. eps15, a novel tyrosine kinase substrate, exhibits transforming activity. Mol. Cell. Biol. 13:58145828.[Abstract]
Field, S.J., R.S. Johnson, R.M. Mortensen, V.E. Papaioannou, B.M. Spiegelman, and M.E. Greenberg. 1992. Growth and differentiation of embryonic stem cells that lack an intact c-fos gene. Proc. Natl. Acad. Sci. USA. 89:93069310.[Abstract]
Formstecher, E., J.W. Ramos, M. Fauquet, D.A. Calderwood, J.-C. Hsieh, B. Canton, X.-T. Nguyen, J.-V. Varnier, J. Camonis, M.H. Ginsberg, and H. Chneiweiss. 2001. PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Dev. Cell. 1:239250.[Medline]
Gille, H., A.D. Sharrocks, and P.E. Shaw. 1992. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature. 358:414424.[CrossRef][Medline]
Hochholdinger, F., G. Baier, A. Nogalo, B. Bauer, H.H. Grunicke, and F. Uberall. 1999. Novel membrane-targeted ERK1 and ERK2 chimeras which act as dominant negative, isotype-specific mitogen-activated protein kinase inhibitors of Ras-Raf-mediated transcriptional activation of c-fos in NIH 3T3 cells. Mol. Cell. Biol. 19:80528056.
Keyse, S.M. 2000. Protein phosphatases, the regulation of mitogen-activated protein kinase signalling. Curr. Opin. Cell Biol. 12:186192.[CrossRef][Medline]
Khokhlatchev, A.V., B. Canagarajah, J. Wilsbacher, M. Robinson, M. Atkinson, E. Goldsmith, and M.H. Cobb. 1998. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell. 93:605615.[Medline]
Kim-Kaneyama, J., K. Nose, and M. Shibanuma. 2000. Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts. J. Biol. Chem. 275:2068520692.
Koutsourakis, M., A. Langeveld, R. Patient, R. Beddington, and F. Grosveld. 1999. The transcription factor GATA6 is essential for early extraembryonic development. Development. 126:723732.
Kurki, P., A. Laasonen, E.M. Tan, and E. Lehtonen. 1989. Cell proliferation and expression of cytokeratin filaments in F9 embryonal carcinoma cells. Development. 106:635640.[Abstract]
Kyriakis, J.M., P. Banerjee, E. Nikolakaki, T. Dai, E.A. Rubie, M.F. Ahmad, J. Avruch, and J.R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 369:156160.[CrossRef][Medline]
Lockett, T.J., and M.J. Sleigh. 1987. Oncogene expression in differentiating F9 mouse embryonal carcinoma cells. Exp. Cell Res. 173:370378.[Medline]
Marais, R., J. Wynne, and R. Treisman. 1993. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 65:663675.
Matsubayashi, Y., M. Fukuda, and E. Nishida. 2001. Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells. J. Biol. Chem. 276:4175541760.
Morrisey, E.E., Z. Tang, K. Sigrist, M.M. Lu, F. Jian, H.S. Ip, and M.S. Parmacek. 1998. GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev. 12:35703590.
Morrisey, E.E., S. Musco, M.Y. Chen, M.M. Lu, J.M. Leiden, and M.S. Parmacek. 2000. The gene encoding the mitogen-responsive phosphoprotein Dab2 is differentially regulated by GATA-6 and GATA-4 in the visceral endoderm. J. Biol. Chem. 275:1994919954.
O'Shea, K.S. 2001. Directed differentiation of embryonic stem cells: genetic and epigenetic methods. Wound Repair Regen. 9:443459.[CrossRef][Medline]
Osborn, M., and K. Weber. 1982. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 24:97132.[Medline]
Pouyssegur, J., V. Volmat, and P. Lenormand. 2002. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem. Pharmacol. 64:755763.[CrossRef][Medline]
Reska, A.A., R. Seger, C.D. Diltz, E.G. Krebs, and E.H. Fischer. 1995. Association of mitogen-activated protein kinase with the microtubule cytoskeleton. Proc. Natl. Acad. Sci. USA. 92:88818885.[Abstract]
Robertson, E.J. 1987. Embryo-derived stem cell lines. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson and R.J. Robertson, editors. IRL Press Limited, Oxford, UK. 71112.
Robinson, M.J., and M.H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9:180186.[CrossRef][Medline]
Rohwedel, J., K. Guan, and A.M. Wobus. 1999. Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs. 165:190202.[CrossRef][Medline]
Saez, E., S.E. Rutberg, E. Mueller, H. Oppenheim, J. Smoluk, S.H. Yuspa, and B.M. Spiegelman. 1995. c-fos is required for malignant progression of skin tumors. Cell. 82:721732.[Medline]
Sejersen, T., J. Sumegi, and N.R. Ringertz. 1985. Expression of cellular oncogenes in teratoma-derived cell lines. Exp. Cell Res. 160:1930.[Medline]
Sgambato, V., P. Vanhoutte, C. Pages, M. Rogard, R. Hipskind, M.-J. Besson, and J. Caboche. 1998. In vivo expression and regulation of Elk-1, a target of the extracellular-regulated kinase signaling pathway, in the adult rat brain. J. Neurosci. 18:214226.
Sherman, M.I., and R.A. Miller. 1978. F9 embryonal carcinoma cells can differentiate into endoderm-like cells. Dev. Biol. 63:2734.[Medline]
Smedberg, J.L., E.R. Smith, C.D. Capo-chichi, A. Frolov, D.-H. Yang, A.K. Godwin, and X.-X. Xu. 2002. Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells. J. Biol. Chem. 277:4091140918.
Smith, E.R., C.D. Capo-chichi, J. He, J.L. Smedberg, D.-H. Yang, A.H. Prowse, A.K. Godwin, T.C. Hamilton, and X.-X. Xu. 2001a. Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells. J. Biol. Chem. 276:4730347310.
Smith, E.R., J.L. Smedberg, M.E. Rula, T.C. Hamilton, and X.-X. Xu. 2001b. Disassociation of MAPK activation and c-Fos expression in F9 embryonic carcinoma cells following retinoic acid-induced endoderm differentiation. J. Biol. Chem. 276:3209432100.
Tresini, M., A. Lorenzini, L. Frisoni, R.G. Allen, and V.J. Cristofalo. 2001. Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast. Exp. Cell Res. 269:287300.[CrossRef][Medline]
Vanhoutte, P., J.L. Nissen, B. Brugg, B.D. Gaspera, M.-J. Besson, R.A. Hipskind, and J. Caboche. 2001. Opposing roles of Elk-1 and its brain-specific isoform, short Elk-1, in nerve growth factor-induced PC12 differentiation. J. Biol. Chem. 276:51895196.
Vasios, G.W., J.D. Gold, M. Petkovich, P. Chambon, and L.J. Gudas. 1989. A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene. Proc. Natl. Acad. Sci. USA. 86:90999103.[Abstract]
Volmat, V., M. Camps, S. Arkinstall, J. Pouyssegur, and P. Lenormand. 2001. The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases. J. Cell Sci. 114:34333443.
Whitehurst, A.W., J.L. Wilsbacher, Y. You, K. Luby-Phelps, M.S. Moore, and M.H. Cobb. 2002. ERK2 enters the nucleus by a carrier-independent mechanism. Proc. Natl. Acad. Sci. USA. 99:74917501.
Yang, S.H., P. Shore, N. Willingham, J.H. Lakey, and A.D. Sharrocks. 1999. The mechanism of phosphorylation-inducible activation of the ETS-domain transcription factor Elk-1. EMBO J. 18:56665674.
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