From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
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ABSTRACT |
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The Mek1 dual specificity protein kinase phosphorylates and activates the mitogen-activated protein kinases Erk1 and Erk2 in response to mitogenic stimulation. The molecular events downstream of Mek and Erk necessary to promote cell cycle entry are largely undefined. In order to study signals emanating from Mek independent of upstream proteins capable of activating multiple signaling pathways, we fused the hormone-binding domain of the estrogen receptor (ER) to the C terminus of constitutively activated Mek1 phosphorylation site mutants. Although 4-OH-tamoxifen stimulation of NIH-3T3 cells expressing constitutively activated Mek-ER resulted in only a small increase in specific activity of the fusion protein, a 5-10 fold increase in total cellular Mek activity was observed over a period of 1-2 days due to an accumulation of fusion protein. Induction of constitutively activated Mek-ER in NIH-3T3 cells resulted in accelerated S phase entry, proliferation in low serum, morphological transformation, and anchorage independent growth. Endogenous Erk1 and Erk2 were phosphorylated with kinetics similar to the elevation of Mek-ER activity. However, elevated Mek-ER activity attenuated subsequent stimulation of Erk1 and Erk2 by serum. 4-OH-tamoxifen stimulation of Mek-ER-expressing fibroblasts also resulted in up-regulation of cyclin D1 expression and down-regulation of p27Kip1 expression, establishing a direct link between Mek1 and the cell cycle machinery.
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INTRODUCTION |
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The transduction of mitogenic signals from the cell membrane to the nucleus involves a cascade of protein binding events and modifications, including a series of phosphorylations resulting in the successive activation of several protein kinases. The intensively studied Ras-MAP1 kinase pathway exemplifies these signaling cascades. Activation of growth factor receptors stimulates nucleotide exchange on the Ras low molecular weight GTP-binding protein (1-3), which then participates in activation of the Raf-1 family of serine/threonine kinases (4). Activated Raf phosphorylates and activates the Mek1 and Mek2 dual specificity kinases, shown to be responsible for phosphorylating the MAP kinases Erk1 and Erk2 on threonine and tyrosine, thus activating them in response to mitogenic stimulation (5-7).
Stimulation of the Ras-MAP kinase pathway ultimately leads to cell proliferation. Ras transformation has been linked to the cell cycle machinery by elevation of cyclin D1 levels in G1, concomitant with down-regulation of the cyclin-dependent kinase inhibitor p27Kip1 (8, 9). It has been shown that overexpression of D-type cyclins can accelerate G1 and contribute to fibroblast transformation (10, 11). Consistent with these results, the requirement for Ras function in induction of cell proliferation in response to mitogenic signaling can be obviated by overexpression of cyclin D1 (9). Elevated cyclin D1 levels were observed in fibroblasts expressing activated c-Raf-1 (12, 13), as well as constitutively activated Mek1.2 However, the molecular events that lead to elevated levels of cyclin D following Erk activation remain murky.
Mek1 and Mek2 (14-16), which share 81% identity, consist almost entirely of a conserved kinase domain flanked by short sequences of lesser homology. Raf-1 activates Mek1 and Mek2 by phosphorylation of 2 serine residues, amino acids 218 and 222 in Mek1 (17-20). Replacement of these two serines with acidic residues results in constitutive activation of Mek enzymatic activity, and overexpression of constitutively active phosphorylation site mutants results in fibroblast transformation (21-26). Phosphorylation of Erk1 and Erk2 on threonine and tyrosine residues in the conserved sequence "TEY" located in the Erk catalytic domain (27, 28) accompanies Mek-induced transformation in most but not all (26) systems.
One limitation of the studies done thus far is that they involve constitutive expression of the activated forms of Mek1. Prolonged passage of cells in the presence of a growth- and transformation-promoting protein raises the issues of autocrine loops, accumulated growth-promoting mutations, and acclimation to the transformed milieu, complicating interpretation of results. In an attempt to circumvent some of these problems, we explored a system that permits the regulation of activated Mek1 expression. A modified estrogen receptor hormone-binding domain (ER HBD) containing a point mutation rendering the HBD unable to bind estrogen while retaining affinity for the synthetic ligand 4-hydroxytamoxifen (4-OH-tamoxifen) has been used to conditionally regulate heterologous proteins (29, 30). In this study, the mutated ER HBD was fused to the C terminus of constitutively activated forms of Mek1 to facilitate investigation of early events in cell proliferation initiated by Mek1 in isolation of parallel pathways that may be activated by extracellular mitogens.
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MATERIALS AND METHODS |
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DNA Construction-- 200 nucleotides from the 5' end of the cDNA encoding the hormone-binding domain (HBD) of the murine estrogen receptor (ER) containing the "tamoxifen mutant" (TM) point mutation (30) was amplified by polymerase chain reaction using the following primers: 5'-AATATGCTAGCATGGGTGCTTCAGGAGACA-3' and 5'-CACACAGTCGACGGGTCTAGAAGGATCATA-3', introducing novel NheI, SalI, and XbaI sites, underlined in that order. 140 nucleotides from the 3' end of the cDNA encoding wild-type murine Mek1 in SK+Mek 4-3A (5) were amplified by polymerase chain reaction using the following primers: 5'-AACCCTGCAGAGAGAGCA-3' and 5'-TTATCGAT GCTAGCTCCGATGCTGGCAGCGTGGGT-3', introducing novel PstI, ClaI, and NheI sites, underlined in that order. The 140-base pair (bp) Mek1 fragment was digested with PstI and ClaI and inserted into Bluescript SK-(Stratagene). This construct was digested with NheI and SalI, into which the NheI-SalI-cut 200-bp fragment of the ER HBD was subcloned, resulting in the insertion of Gly-Ala-Ser between Ile392 of Mek1 and Met284 of the ER HBD. This construct was confirmed by sequence analysis and digested with SmaI and PstI, into which the N-terminal 300-bp blunt/PstI fragment of MekI was inserted, followed by digestion with PstI and insertion of the DS (Asp218) or DD (Asp218, Asp222) Mek1 phosphorylation site mutant (23) 700-bp PstI fragments. Restriction sites between the polylinker SpeI and NotI were removed, and the resulting construct was cut with SalI and XbaI, into which site the 3' 800-bp of the ER HBD (SalI-XbaI fragment) was inserted. Finally, the entire cDNAs encoding the Mek1-ER fusions were cut out of Bluescript with BamHI and SalI and inserted into BamHI-SalI-digested pBabe-puro.
Cell Culture and Transfection--
NIH-3T3 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin.
Calcium phosphate transfections were done using a buffer kit from 5 Prime 3 Prime, Inc., Boulder, CO. Transfected cells were selected
in 2 µg/ml puromycin (Sigma) for 12 days, and individual
drug-resistant colonies were isolated. Fusion constructs were induced
using 100 nM 4-hydroxy-tamoxifen (Research Biochemicals),
typically for 24 h. Unless otherwise noted, cell count experiments
were performed by seeding 6-cm plates with 6.5 × 104
cells in DMEM supplemented with 10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin, incubating for 2 days, replacing medium with that containing 0.5% calf serum with or without
100 nM 4-OH-tamoxifen, then counting and/or photographing cells over the subsequent 3 days.
Flow Cytometry-- Cells were placed in medium containing 0.5% calf serum simultaneously with addition of 100 nM 4-OH-tamoxifen. 24 h later, cells were trypsinized and resuspended in 100 µl of cold phosphate-buffered saline containing 0.1% dextrose, to which 3 ml of 70% ethanol was added, incubated on ice for 30 min, and spun down. Pellets were resuspended in 40 mM sodium citrate, pH 7.4, containing 70 µM propidium iodide and 100 µg/ml RNase, incubated at 37 °C for 30 min, and analyzed by fluorescence-activated cell sorting using the Cellquest program (Becton Dickinson).
Immunoblotting-- Whole cell lysates or immunoprecipitates separated by SDS-polyacrylamide gel electrophoresis were electrophoretically transferred to polyvinylidene difluoride membranes, blocked in Tris-buffered saline (20 mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Tween 20 and 5% bovine serum albumin, probed with the relevant antibody (all secondary antibody incubations were done in Tris-buffered saline containing 0.1% Tween 20 and 5% milk), and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). Mek-ER fusion proteins were detected with monoclonal anti-ER Ab-1 (Oncogene Research/Calbiochem) or monoclonal anti-Mek1 3D9 (Zymed); endogenous Mek1 was detected with monoclonal anti-Mek1 3D9 (Zymed). Activation-specific phosphorylated species of Erk1 and Erk2 were detected with polyclonal phospho-specific Erk (New England Biolabs), cyclin D1 with monoclonal anti-cyclin D1 72-13G (Santa Cruz), and Kip1 with monoclonal anti-p27 Kip1 (Transduction Laboratories).
Immunoprecipitation-- Mek-ER fusion proteins were immunoprecipitated from lysates prepared as described above with anti-ER Ab-1 (Oncogene Research/Calbiochem) and protein G-agarose (Zymed or Santa Cruz).
Immunocomplex Kinase Assays--
Mek-ER fusion protein kinase
activity was measured as follows. Anti-ER immunoprecipitates (generally
from 100 µg of cell lysate) were washed twice with lysis buffer and
once with kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin) and
split into two tubes. Half of each immunoprecipitate was used for
immunoblotting, and half of each immunoprecipitate was incubated at
30 °C for 15 min in 20 µl of kinase buffer containing 10 µCi
[-32P]ATP, 25 µM cold ATP, and 1 µg
GST-Erk1 K63M per sample. Reactions were terminated by addition of an
equal volume of SDS sample buffer containing 50 mM EDTA,
boiled for 5 min, and separated by 8% SDS-polyacrylamide gel
electrophoresis. Gels were Coomassie-stained, dried, and exposed to
film. Gel sections containing Coomassie-stained substrate and autophosphorylated fusion protein were quantitated by scintillation counting.
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RESULTS |
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Biochemical Analysis of Fibroblasts Expressing Mek1-ER Fusion Proteins-- The constitutively activated Mek1 phosphorylation site mutants Asp218 (DS) and Asp218,Asp222 (DD) exhibit elevated kinase activity toward kinase-inactive GST-Erk1 and transform fibroblasts in tissue culture (23, 26). The DD form is more active and more transforming than Mek1-DS, consistent with the introduction of a constitutive negative charge at the two sites required to be phosphorylated for full activation of wild-type Mek1. In order to conditionally regulate activity of these Mek1 phosphorylation site mutants, fusion constructs were engineered in which the coding sequence for the TM mutant (30) of the estrogen receptor hormone-binding domain (ER HBD) was placed in frame at the 3' end of the Mek1 cDNAs (construction described under "Materials and Methods"). These fusion constructs were then inserted into the expression vector pBabe-puro, creating pBp Mek1-DSER and pBp Mek1-DDER. NIH-3T3 cells were transfected with pBp Mek1-DSER, pBp Mek1-DDER, or empty pBp vector and selected in puromycin. Stable clonal lines were isolated and screened for basal levels of fusion protein expression by whole cell lysate immunoblot analysis using an anti-ER antibody (data not shown). Representative lines exhibiting high levels of expression were used in the following experiments.
Cells expressing empty vector, Mek1-DSER or Mek1-DDER, were treated for 24 h with 100 nM 4-OH-tamoxifen and lysed. Fusion protein kinase activity was measured in the absence of endogenous Mek1 by anti-ER immune complex kinase assay, using kinase inactive GST-Erk1 K63M as a substrate (Fig. 1A). A 5-10-fold increase in fusion protein kinase activity toward kinase-inactive GST-Erk1 was observed after tamoxifen treatment; in the experiment shown, Mek1-DSER activity was increased 7-fold, and Mek1-DDER activity was increased 6.5-fold. A concomitant increase in fusion protein autophosphorylation was also observed. Treatment with an equal volume of ethanol vehicle had no effect on fusion protein kinase activity (data not shown). Half of each immunoprecipitate used in the kinase assay in Fig. 1A was immunoblotted with anti-ER to detect protein levels in each immunoprecipitate. Significantly, the 4-OH-tamoxifen-stimulated lysates exhibited higher levels of precipitable fusion protein contributing to the observed kinase activity (Fig. 1B), suggesting that the increase in observed kinase activity was not due to an increase in specific activity.
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Effects of Induction of Mek-ER on Cell Growth-- The cells were first examined for the ability to proliferate in low serum. 6 × 104 cells transfected with vector or Mek1-DDER were plated and grown for 2 days in complete medium containing 10% calf serum. The cells were then placed in medium containing low (0.5%) calf serum and treated with 100 nM 4-OH-tamoxifen or an equal volume of ethanol vehicle for 24 h and then counted each of the following 3 days. Whereas the number of vector-transfected and unstimulated Mek1-DDER cells peaked at day 3 and then decreased, the number of 4-OH-tamoxifen-stimulated Mek1-DDER cells continued to increase until day 5 (Fig. 3A). At no time during this representative experiment did any of the plates reach confluence, indicating that the inhibition of growth was not due to contact inhibition (Fig. 3B). Stimulation of Mek1-DSER cells with 4-OH-tamoxifen resulted in an intermediate growth phenotype (Fig. 3C), while untreated Mek1-DDER cells grown continuously in 10% calf serum proliferated three times as fast as Mek1-DDER cells treated with 4-OH-tamoxifen but grown in low serum (data not shown).
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Characterization of Fusion Protein-induced Transformation-- Constitutive expression of activated Mek1 phosphorylation site mutants has been previously shown to transform fibroblasts; however, the kinetics of cell transformation by these activated kinases were in some cases delayed, raising the issue of whether secondary effects were involved (21, 22, 25, 26). The ability of activated Mek1-ER to transform cells upon 4-OH-tamoxifen stimulation was therefore examined. When grown in complete medium containing 10% calf serum, the cells expressing Mek1-DDER exhibited morphological alteration and were more refractile within 24 h of hormone addition (Fig. 5A), a time at which significant levels of fusion protein and anti-ER-precipitable kinase activity have accumulated (Fig. 2, A and B). No morphological alterations were observed in the Mek1-DSER expressing cells after 24 h 4-OH-tamoxifen stimulation (Fig. 5A), consistent with Mek1-DSER activity in these cells at this time point, 2-fold greater than that of unstimulated Mek1-DDER (data not shown). However, prolonged treatment of Mek1-DSER-expressing NIH-3T3 cells with 100 nM 4-OH-tamoxifen did result in detectable morphological changes, although not to the extent observed in Mek1-DDER-expressing NIH-3T3 cells (Fig. 5B). Mek1-DDER cells exhibited a similar change in morphology when placed in medium containing low serum simultaneously with 4-OH-tamoxifen addition, in stark contrast to the flattened, quiesced control cells (Fig. 3B).
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Fusion Protein Induction of Erk Phosphorylation-- One potential advantage of this inducible system is in examination of the kinetics of downstream signaling protein activation in response to induction of activated Mek. Activation of the known substrates of Mek1, the MAP kinases Erk1 and Erk2 (5-7), was first examined. As expected, time course analysis of activation-specific Erk phosphorylation indicated that the appearance of phosphorylated endogenous Erk2 coincided with accumulation of Mek1-DDER, approximately 6 h after stimulation with 4-OH-tamoxifen (Fig. 6A). Erk2 activation-specific phosphorylation peaked 2 days after hormone stimulation of Mek1-DDER and remained elevated for at least 12 weeks, the last time point examined (Fig. 6B and data not shown). This is in contrast to results from a set of cell lines constitutively expressing activated Mek1-DD, in which basal levels of endogenous Erk activity are comparable to levels in vector-transfected cells (26). Induction of Mek1-DSER resulted in only a slight increase in Erk2 phosphorylation after 2 days of hormone treatment. Erk1 phosphorylation is less sensitive in this assay and is only observed when exceptionally high levels of Erk2 phosphorylation are present (such as the last lane in the middle panel of Fig. 6B).
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Analysis of Cell Cycle Proteins-- Effects of Ras pathway activation on cell cycle components involved in G1 progression have recently been described (8, 9, 12, 13, 31). D-type cyclins are responsive to growth factor levels and are required for S phase entry (35, 36). Because we observed an increase in the S phase population of Mek1-DDER cells following stimulation with 4-OH-tamoxifen (Fig. 4), we examined whether Mek1-DDER affects proteins involved in cell cycle entry. Anti-cyclin D1 immunoblot analysis revealed that induction of Mek1-DDER but not Mek1-DSER caused an elevation of cyclin D1 protein after 2 days 4-OH-tamoxifen treatment (Fig. 8A). Cyclin D1 levels remained elevated upon prolonged treatment of the Mek1-DDER cells with 4-OH-tamoxifen, whereas no detectable elevation of cyclin D1 levels was observed after prolonged treatment of the Mek1-DSER cells (Fig. 8B).
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DISCUSSION |
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We have utilized an inducible system to study the downstream effects of Mek activation and its role in stimulating cell growth and transformation. We focused our attention on Mek1 mutants that are constitutively active as the result of substitution of aspartate residues for serines normally phosphorylated by activated Raf. We reasoned that fusion of such mutants to the hormone-binding domain of the murine estrogen receptor would permit rapid induction of the active kinases upon treatment with 4-OH-tamoxifen. In this study only a small (1.5-fold) immediate increase in specific activity was obtained by 4-OH-tamoxifen treatment of fibroblasts expressing these fusion constructs. This small elevation of Mek1 activity did not produce any detectable change in cellular phenotype at early time points (Fig. 6A and data not shown). Within 24 h, however, an increase in fusion protein levels was observed, accompanied by a proportional increase in total cellular anti-ER-precipitable kinase activity (Fig. 1). This elevation of Mek1-ER kinase activity permitted the initiation of studies to evaluate the molecular mechanisms by which Mek1 influences the cell cycle.
24 h after 4-OH-tamoxifen addition, fusion protein levels were 3-5-fold greater than that of endogenous Mek1 (Fig. 1D), comparable to levels that have been previously described to transform fibroblasts when constitutively expressed (26). Hormone stimulation of NIH-3T3 cells expressing the doubly substituted Mek1-DDER caused morphological transformation within 24 h (Fig. 5A). Elevation of fusion protein levels and protein kinase activity was detected 6-12 h after initiation of treatment. Thus, the time necessary for Mek1 to elicit cell transformation events is no greater than 12-18 h. Stimulated Mek1-DDER cells can also grow in an anchorage-independent manner (Table I), but the kinetics of this phenotype are more difficult to analyze for technical reasons. Induction of singly substituted Mek1-DSER is only weakly transforming (Fig. 5, A and B, and Table I), consistent with its poor ability to activate Erk1 and Erk2, compared with that of Mek1-DDER (Fig. 6B).
The ability of the Mek1-DDER expressing NIH-3T3 cells to grow in low serum upon treatment with 4-OH-tamoxifen provides a further measure of a transformed phenotype (Fig. 3). As in the previous assays, Mek1-DSER induction caused only a weak cell growth response compared with Mek1-DDER. It should be noted that untreated Mek1-DDER cells maintained in complete medium containing 10% calf serum grew three times as fast as the Mek1-DDER cells placed in low serum containing 4-OH-tamoxifen (data not shown), indicating that Mek1 activation cannot completely substitute for the growth factors provided by serum. Serum may act in two ways to cause a greater response as follows: it may stimulate phosphorylation of endogenous Mek1 on both serines 218 and 222, which apparently activates Mek1 more efficiently than substitution of these two residues with aspartate (48 and Fig. 7A); or it may activate other pathways in parallel to Mek1 signaling that synergize to promote further cell growth and transformation. It is therefore unlikely that Mek1 activation is sufficient for maximal stimulation of cell growth. However, the question of whether all of the signals emanating from activated Raf are dependent on Mek1 and/or Mek2 remains: is Mek1 activation functionally equivalent to Raf activation in fibroblast transformation?
Another issue is whether Mek1 activation is sufficient for cell cycle entry or oncogenic transformation. It has been reported that heparin-binding epidermal growth factor expression is induced by activated c-Raf-1 or Ras and activates the Jnk pathway in the process of stimulating proliferation (12, 42). Although the kinetics of Erk phosphorylation between 24 and 48 h (Fig. 6B) do not correlate in a linear fashion, implicating either delayed activation of another kinase capable of phosphorylating Erk or an autocrine loop that becomes fully activated only after 24 h of 4-OH-tamoxifen treatment, we detected no activation of Jnk1 (data not shown). The kinetics of both S phase entry and cell transformation are relatively rapid, as described above, and induction of Mek1-DDER causes up-regulation of cyclin D1 and down-regulation of p27Kip1, two hallmarks of G1 progression (Fig. 8). Although one could argue that the cells in the experiment shown in Fig. 3A were still cycling at the time of hormone addition, this experiment has been repeated with cells placed in low serum 24 h prior to 4-OH-tamoxifen stimulation; the stimulated Mek1-DDER cells still continued to proliferate (data not shown). These data are consistent with the hypothesis that activation of Mek1 is sufficient to drive cell cycle progression.
Serum stimulation of endogenous Erk phosphorylation is attenuated in cells expressing elevated levels of Mek1-DDER (Fig. 7, lanes 4, 8, and 12), which is consistent with previously reported results which demonstrated a delay or attenuation of serum stimulation of Erk activity in oncogene-transformed cells (43-45). Although constitutively activated Mek1-DDER is expressed for the duration of the serum stimulation, any serum-driven increase in Erk activity in the Mek1-DDER cells would have had to be mediated by endogenous Mek, as the activity of Mek1-DDER is not affected by serum. Northern analysis for the expression of MKP-1, a dual specificity phosphatase normally induced by serum and reported to inactivate Erk1 and Erk2 (46), revealed that MKP-1 message levels are not stimulated by serum in Mek1-DDER expressing fibroblasts (data not shown). The mechanism of attenuation could therefore involve another Erk-specific phosphatase, a protein that prevents interaction of Mek and Erk, or a feedback mechanism that inhibits an upstream component of the signal transduction pathway leading to activation of endogenous Mek in response to serum stimulation.
Modulation of cell cycle components was also observed in response to induction of constitutively activated Mek1-ER. Two events associated with G1 progression, up-regulation of cyclin D1 levels and down-regulation of p27Kip1 levels (35, 36, 39-41), were shown to occur within 24 h of treatment with 4-OH-tamoxifen (Fig. 8). High level induction of activated c-Raf-1 or activated Ras has been demonstrated to up-regulate expression of the cyclin-dependent kinase inhibitor p21Cip1, accompanied by cell cycle arrest (13, 47, 48). We did not observe cell cycle arrest in response to hormone induction of Mek1-DDER (Fig. 3), although it is possible that the levels of activity attained were not sufficient to induce p21Cip1. Future studies will focus on the influence of activated Mek1 on p21Cip1 expression, the question of the respective effects of Mek1 and c-Raf-1 on the cell cycle, and the role of feedback control in this pathway.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Klagsbrun for the heparin-binding epidermal growth factor cDNA. We also thank Jonathan Samuel for assistance with the Jnk assays and Dr. Sheng Ma and Dr. Barbara Brott for critical reading of the manuscript.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant CA42580 and National Institutes of Health Postdoctoral Fellowship 1 F32 GM19098-01 (to H. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular and
Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA
02138. Tel.: 617-495-9686; Fax: 617-495-0681; E-mail: heidi{at}biosun.harvard.edu.
§ Holds the John F. Drum American Cancer Society Research Professorship.
1 The abbreviations used are: MAP, mitogen-activated protein; ER, estrogen receptor; HBD, hormone-binding domain; GST, glutathione S-transferase; bp, base pair; Mek, MAP kinase/extracellular signal-regulated kinase kinase; TM, tamoxifen mutant.
2 Q. Hou and R. Erikson, unpublished data.
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REFERENCES |
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