An Analysis of Mek1 Signaling in Cell Proliferation and Transformation
1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês
10.1074/jbc.273.21.13280
ISSN1083-351X
AutoresHeidi Greulich, Raymond L. Erikson,
Tópico(s)Cell death mechanisms and regulation
ResumoThe 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. 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. 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-MAP 1The 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. kinase pathway exemplifies these signaling cascades. Activation of growth factor receptors stimulates nucleotide exchange on the Ras low molecular weight GTP-binding protein (1McCormick F. Nature. 1993; 363: 15-16Crossref PubMed Scopus (445) Google Scholar, 2Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar, 3Sasaoka T. Langlois W.J. Leitner J.W. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 32621-32625Abstract Full Text PDF PubMed Google Scholar), which then participates in activation of the Raf-1 family of serine/threonine kinases (4Avruch J. Zhang X. Kyriakis J. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (542) Google Scholar). 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 (5Crews C. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (740) Google Scholar, 6Ashworth A. Nakielny S. Cohen P. Marshall C. Oncogene. 1992; 7: 2555-2556PubMed Google Scholar, 7Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T.A.J. Michel H. Hunt D.F. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (119) Google Scholar). 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 (8Liu J.-J. Chao J.-R. Jiang M.-C. Ng S.-Y. Yen J.J.-Y. Yang-Yen H.-F. Mol. Cell. Biol. 1995; 15: 3654-3663Crossref PubMed Scopus (263) Google Scholar, 9Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (372) Google Scholar). It has been shown that overexpression of D-type cyclins can accelerate G1 and contribute to fibroblast transformation (10Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar, 11Hinds P.W. Dowdy S.F. Eaton E.N. Arnold A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 709-713Crossref PubMed Scopus (455) Google Scholar). 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 (9Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (372) Google Scholar). Elevated cyclin D1 levels were observed in fibroblasts expressing activated c-Raf-1 (12Kerkhoff E. Rapp U.R. Mol. Cell. Biol. 1997; 17: 2576-2586Crossref PubMed Scopus (152) Google Scholar, 13Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar), as well as constitutively activated Mek1. 2Q. Hou and R. Erikson, unpublished data. However, the molecular events that lead to elevated levels of cyclin D following Erk activation remain murky. Mek1 and Mek2 (14Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar, 15Wu J. Harrison J.K. Dent P. Lynch K.R. Weber M.J. Sturgill T.W. Mol. Cell. Biol. 1993; 13: 4539-4548Crossref PubMed Scopus (125) Google Scholar, 16Brott B.K. Alessandrini A. Largaespada D.A. Copeland N. Jenkins N.A. Crews C. Erikson R.L. Cell Growth Differ. 1993; 4: 921-929PubMed Google Scholar), 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 (17Kyriakis J.M. Force T.L. Rapp U.R. Bonventre J.V. Avruch J. J. Biol. Chem. 1993; 268: 16009-16019Abstract Full Text PDF PubMed Google Scholar, 18Huang W. Alessandrini A. Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10947-10951Crossref PubMed Scopus (103) Google Scholar, 19Zheng C.-F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (302) Google Scholar, 20Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (468) Google Scholar). 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 (21Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar, 22Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1263) Google Scholar, 23Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar, 24Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar, 25Brunet A. Pages G. Pouyssegur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar, 26Alessandrini A. Greulich H. Huang W. Erikson R.L. J. Biol. Chem. 1996; 271: 31612-31618Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Phosphorylation of Erk1 and Erk2 on threonine and tyrosine residues in the conserved sequence "TEY" located in the Erk catalytic domain (27Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.-H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (842) Google Scholar, 28Alessandrini A. Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8200-8204Crossref PubMed Scopus (78) Google Scholar) accompanies Mek-induced transformation in most but not all (26Alessandrini A. Greulich H. Huang W. Erikson R.L. J. Biol. Chem. 1996; 271: 31612-31618Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) 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 (29Danielian P. White R. Hoare S. Fawell S. Parker M. Mol. Endocrinol. 1993; 7: 232-240Crossref PubMed Scopus (1) Google Scholar, 30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (704) Google Scholar). 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. 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 (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (704) Google Scholar) 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 (5Crews C. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (740) Google Scholar) 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 withNheI and SalI, into which theNheI-SalI-cut 200-bp fragment of the ER HBD was subcloned, resulting in the insertion of Gly-Ala-Ser between Ile392of Mek1 and Met284 of the ER HBD. This construct was confirmed by sequence analysis and digested withSmaI 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 (23Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar) 700-bp PstI fragments. Restriction sites between the polylinker SpeI andNotI were removed, and the resulting construct was cut withSalI 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. 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 × 104cells 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. Colony formation was assayed by suspending 5 × 105cells in DMEM containing 10% calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 nm tamoxifen, and 0.25% agar, plated on a layer of DMEM containing 10% calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.5% agar in 6-cm tissue culture plates. Plates were incubated at 37 °C for 2 weeks, and then colonies were arbitrarily divided into small (<0.5 mm) or large (≥0.5 mm) categories, and colonies in 5 randomly chosen 1-cm2areas were counted. Cells were lysed in a buffer containing 10 mm Tris, pH 7.6, 1% Triton X-100, 50 mm NaCl, 30 mmNaPPi, 50 mm NaF, 1 mm EGTA, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, and 20 μg/ml aprotinin. Protein concentrations were measured by the Bradford assay using the Bio-Rad reagent. Cells were placed in medium containing 0.5% calf serum simultaneously with addition of 100 nm4-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 mmsodium 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). 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). 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). 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 [γ-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. 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 (23Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar, 26Alessandrini A. Greulich H. Huang W. Erikson R.L. J. Biol. Chem. 1996; 271: 31612-31618Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (704) Google Scholar) 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. 1 A). 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. 1 A 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. 1 B), suggesting that the increase in observed kinase activity was not due to an increase in specific activity. The increase in fusion protein expression was confirmed by whole cell lysate immunoblot analysis (Fig. 1 C). Elevation of ER fusion protein levels in response to hormone induction, possibly due to protein stabilization, has been previously described (31Jackson P. Baltimore D. Picard D. EMBO J. 1993; 12: 2809-2819Crossref PubMed Scopus (45) Google Scholar, 32Samuels M.L. Weber M. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (323) Google Scholar). Because the expression levels of Mek1-DDER are slightly higher than those of Mek1-DSER, the experiment was repeated normalizing for the amount of fusion protein in each immunoprecipitate. The resulting basal Mek1-DDER activity was 4-fold higher than basal Mek1-DSER activity, whereas 4-OH-tamoxifen-stimulated Mek1-DDER activity was 2.5-fold higher than stimulated Mek1-DSER activity (data not shown). Basal and induced levels of fusion protein expression were also compared with that of endogenous Mek1 by whole cell lysate anti-Mek1 immunoblot analysis (Fig. 1 D). Whereas the uninduced Mek1-DSER and Mek1-DDER proteins were expressed at only about 10% the level of endogenous Mek1 protein, 24–48 h of tamoxifen stimulation led to an increase in fusion protein expression to levels 3–5-fold greater than that of endogenous Mek1, comparable to levels in previously described fibroblasts constitutively expressing these activated Mek1 phosphorylation site mutants (26Alessandrini A. Greulich H. Huang W. Erikson R.L. J. Biol. Chem. 1996; 271: 31612-31618Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The kinetics of induction of fusion protein levels and activity upon addition of 100 nm 4-OH-tamoxifen were then examined. No large increases in kinase activity were observed until 6–12 h after hormone addition (Fig. 2 A), at which time the levels of fusion protein began to increase as well (Fig.2 B). However, a slight but reproducible increase in specific activity, about 1.5-fold, was detected as early as 15 min after 4-OH-tamoxifen stimulation (data not shown). Thus, unlike previous reports using ER HBD fusion proteins (12Kerkhoff E. Rapp U.R. Mol. Cell. Biol. 1997; 17: 2576-2586Crossref PubMed Scopus (152) Google Scholar, 32Samuels M.L. Weber M. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (323) Google Scholar), we observed only a barely detectable immediate increase in fusion protein-specific activity, followed by a more robust delayed response due to fusion protein accumulation. The levels of fusion protein induced by 4-OH-tamoxifen addition peaked 48 h after hormone addition and remained elevated for at least 12 weeks, the latest time point tested (Fig. 2 C and data not shown). Anti-ER-precipitable kinase activity remained elevated as well (Fig. 2 D and data not shown). 4-OH-tamoxifen titration indicated that maximal stimulation of fusion protein expression and kinase activity occurred between 50 and 100 nm (data not shown). Subsequent experiments therefore involved stimulation with 100 nm 4-OH-tamoxifen unless otherwise noted. Removal of 4-OH-tamoxifen from the medium resulted in complete elimination of the induced fusion protein over a 2-day period, and it should be noted that there was no further elevation of fusion protein levels or kinase activity by repeated addition of hormone once a day for 3 days as compared with a single addition for 3 days (data not shown). 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. 3 A). 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. 3 B). Stimulation of Mek1-DSER cells with 4-OH-tamoxifen resulted in an intermediate growth phenotype (Fig. 3 C), 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). The effects of Mek1-DDER on the cell cycle were analyzed by flow cytometry (Fig. 4). Cells were placed in 0.5% serum and treated with 100 nm 4-OH-tamoxifen or ethanol vehicle for 24 h prior to analysis. Although no significant effect of Mek1-DDER induction on G2/M was detected, a 3–4-fold increase in the percentage of cells in S phase was observed following 4-OH-tamoxifen treatment. The percentage of Mek1-DDER cells in S phase peaked after 18 h of 4-OH-tamoxifen treatment and slowly declined over a period of 48 h (data not shown). 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 (21Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar, 22Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1263) Google Scholar, 25Brunet A. Pages G. Pouyssegur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar, 26Alessandrini A. Greulich H. Huang W. Erikson R.L. J. Biol. Chem. 1996; 271: 31612-31618Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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. 5 A), a time at which significant levels of fusion protein and anti-ER-precipitable kinase activity have accumulated (Fig. 2, A andB). No morphological alterations were observed in the Mek1-DSER expressing cells after 24 h 4-OH-tamoxifen stimulation (Fig. 5 A), 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. 5 B). 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.3 B). The ability of the Mek1-ER expressing cells to grow in an anchorage-independent manner was then analyzed by colony formation in soft agar. NIH-3T3 cells transfected with vector alone, Mek1-DSER, or Mek1-DDER were stimulated for 24 h with 100 nm4-OH-tamoxifen or left unstimulated and were plated in soft agar medium with or without 100 nm 4-OH-tamoxifen. Plates were incubated 12 days, then the number of colonies were counted. No colonies were observed on vector or Mek1-DSER plates in the absence of hormone stimulation (Table I), although a low level of background colony formation was seen on the Mek1-DDER plate, probably due to the basal level of anti-ER-precipitable kinase activity in these cells (Fig. 1 A). The number of colonies on the 4-OH-tamoxifen-stimulated Mek1-DDER plate was several orders of magnitude greater than that on the unstimulated plate. Only a small number of colonies was observed on the 4-OH-tamoxifen-stimulated Mek1-DSER plate, comparable to the unstimulated Mek1-DDER plate.Table IGrowth in soft agar of tamoxifen-treated NIH-3T3 cells expressing Mek-ER fusion proteins 0.5 mmVector01-aNumber of colonies counted in five randomly chosen 1-cm2 fields.0Vector + T1-bT, 100 nm 4-OH-tamoxifen.00DSER00DSER + T40DDER90DDER + T41604801-a Number of colonies counted in five randomly chosen 1-cm2 fields.1-b T, 100 nm 4-OH-tamoxifen. Open table in a new tab 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 (5Crews C. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (740) Google Scholar, 6Ashworth A. Nakielny S. Cohen P. Marshall C. Oncogene. 1992; 7: 2555-2556PubMed Google Scholar, 7Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T.A.J. Michel H. Hunt D.F. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (119) Google Scholar), 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. 6 A). 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. 6 B 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
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